Method for producing cereulide and derivative thereof, intermediate for production of cereulide, and cereulide derivative

ABSTRACT

The purpose of the present invention is to provide a novel method for producing cereulide and a derivative thereof; an intermediate for cereulide; and a novel cereulide derivative. A novel didepsipeptide, a novel tetradepsipeptide, a novel octadepsipeptide and a novel dodecadepsipeptide are prepared. A linear precursor of cereulide or a derivative thereof, which is composed of any one of the novel depsipeptides, is cyclized by forming an intramolecular amide bond.

TECHNICAL FIELD

The present invention relates to a method for producing cereulide and aderivative thereof. The present invention also relates to anintermediate for production of cereulide and a cereulide derivative.

BACKGROUND ART

Bacillus cereus that lives in the soil propagates in starch-based foodssuch as rice, pilaf and spaghetti, and produces cereulide that inducesan emetic action on an animal such as human. It is also known that notall Bacillus cereus species produce cereulide, but only Bacillus cereusspecies which acquire a cereulide synthetic gene produce this toxicsubstance.

Currently, when food poisoning by cereulide is suspected, a method foridentifying cereulide in an extract of the food by HPLC or LC/MS iscarried out. There is also a method for carrying out bioassay usingcells, using a vacuolating action of cereulide as an index.

It is known that such cereulide has high heat resistance, acidresistance, and resistance to digestive enzymes, is not deactivated inthe process of cooking and digestion, and acts on the small intestinenervous system to induce vomiting phenomenon. Furthermore, liver damage,mitochondrial toxicity, induction of alteration of cellular morphology,apoptosis induction and the like are reported, but researches ofmolecular mechanism of the vomiting phenomenon or other toxicity at amolecular level have not made any progress.

For the determination of food poisoning, methods by detection of asynthetic enzyme gene in a specimen (Patent Documents 1 and 2) are alsosuggested.

However, development of a simple analysis method, not a method requiringan expensive analytical instrument and skilled technique, is desirable.

For qualitative or quantitative analysis for determining food poisoningdue to cereulide, as well as toxicity evaluation or elucidation of themechanism at a cellular or molecular level, a pure cereulide referencestandard is often required.

Currently commercially available cereulide is extracted from a culturesolution of Bacillus cereus, and is available as a methanol solution ofcereulide.

As a method for obtaining such cereulide, methods for synthesizingcereulide and a derivative thereof are also suggested (Non-PatentDocuments 1 and 2).

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: WO 3/097821

Patent Document 2: WO 2006-6256

Non-Patent Documents

Non-Patent Document 1: Bioorganic & Medicinal Chemistry Letters, Vol. 5,No. 23, 2855-2858 (1995)

Non-Patent Document 2: Synthesis 2009, No. 13, 2184-2204

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

Currently commercially available cereulide is very expensive, and isderived from a culture solution, thus the purity of the cereulide is notso high. It has been desirable to easily produce highly pure cereulide.

Thus, an object of the present invention is to provide a method forproducing cereulide and a derivative thereof, and a cereulidederivative.

Means for Solving the Problems

The present inventors have intensively studied, and found that theobject can be achieved by the method for producing cereulide and aderivative thereof, an intermediate for production of cereulide, and acereulide derivative, whereby the present invention is accomplished.

More specifically, the present invention relates to cereulide or aprecursor of a derivative thereof, selected from the group consisting ofthe following formulae:

wherein Y represents OH or NH(CH₂)₅COOH.

The present invention further relates to a didepsipeptide represented by

wherein X represents isopropyl, (CH₂)₂COOH or (CH₂)₂CONH(CH₂)₅COOH.

The present invention further relates to a depsipeptide as shown below

wherein l is an integer of 0 to 2, n is an integer of 0 to 2, and m isan integer of 0 or 1, wherein l, m and n are not simultaneously 0, andl+m+n is 2 or less.

The present invention further relates to a method for producingcereulide or a derivative thereof represented by

wherein X represents isopropyl, (CH₂)₂COOH or (CH₂)₂CONH(CH₂)₅COOH,comprising a cyclization reaction by formation of an intramolecularamide bond of the precursor of cereulide or a derivative thereof.

In the production method, it is preferred to further comprise a step ofpreparing the didepsipeptide.

In the production method, it is preferred to further comprise a step ofpreparing the depsipeptide of

The present invention further relates to a cereulide derivativerepresented by the following formula:

wherein R represents (CH₂)₂COOH or (CH₂)₂CONH(CH₂)₅COOH.

Effect of the Invention

According to the method for producing cereulide and a derivative thereofof the present invention, highly pure cereulide or a derivative thereofcan be easily obtained. Furthermore, the cereulide derivative of thepresent invention is useful for antibody preparation for detectingcereulide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ¹H-NMR (CDCl₃) spectrum of the octadepsipeptide of thepresent invention.

FIG. 2 is a ¹H-NMR (CDCl₃) spectrum of the dodecadepsipeptide of thepresent invention.

FIG. 3 is a ¹H-NMR (CDCl₃) spectrum of the dodecadepsipeptide of thepresent invention.

FIG. 4 is a ¹H-NMR (CDCl₃) spectrum of the dodecadepsipeptide of thepresent invention.

FIG. 5 is a ¹H-NMR (CDCl₃) spectrum of the cereulide of the presentinvention.

MODE FOR CARRYING OUT THE INVENTION

Cereulide is a 36-membered macrocyclic compound (macrolide) in which sixamino acids and six hydroxy acids are alternatively linked by an amidebond and an ester bond, and can also be called ascyclododecadepsipeptide. Cereulide has the structure as follows, that isconstituted of two types of amino acids, L-valine (L-Val) and D-alanine(D-Ala) and two types of α-hydroxy acids, D-leucine acid (D-O-Leu) andL-valine acid (L-O-Val).

In many cases, cereulide functions as an ionophore of K⁺, Na⁺, or NH₄ ⁺.Such a capturing action is considered as a cause of bioactivities.

The inventors have considered that the step of cyclizing a linearprecursor to construct a 36-membered ring structure is the mostimportant key stage in the total synthesis of cereulide, and havefocused on that the method by formation of an ester bond isconventionally adopted for achieving this cyclization reaction.Regarding cereulide and a derivative, all the conventionally knownsynthetic methods include a cyclization step as shown in the followingschematic view.

In the present invention, a tetradepsipeptideL-Val-D-O-Leu-D-Ala-L-O-Val is focused as a repeating structure unit ofcereulide. More specifically, cereulide is a macrolide cyclized bylinking three of the repeating structure units, and can also bedescribed as (L-Val-D-O-Leu-D-Ala-L-O-Val)₃.

The present invention relates to a novel production method includingmacrocyclic construction by amide bond formation as a key reaction,unlike a conventional method. The starting materials in the productionmethod of the present invention are all commercially available, and twohydroxy acids, i.e., D-leucine acid (D-O-Leu) and L-valine acid(L-O-Val), can be prepared from commercially available D-leucine andL-valine, by a method known in the literature, and used in the followingfragment synthesis.

Further, in the present invention, taking note on the problem that, inthe depsipeptide synthesis, the longer the peptide chain is, the largerthe steric hindrance is, whereby the solubility in an organic solvent isreduced to cause reduction in the reaction yield. Thus, in order toavoid such a problem, a method of first synthesizing a tetradepsipeptiderepeating structural unit, followed by binding two of the structuralunits to obtain an octadepsipeptide (2×structural unit), thereafterfurther binding the octadepsipeptide with a tetradepsipeptide to preparea linear dodecadepsipeptide of three structural units (precursor) andcyclizing the dodecadepsipeptide is adopted.

The intramolecular cyclization reaction of a precursor by amide bondconstruction in the present invention can also be carried out bychanging various reaction conditions such as a condensing agent, asolvent and the temperature, and can also be optimized for realizing ahigh yield.

According to the method of the present invention, synthesis of cereulideand a derivative thereof can be sought in commercially available rawmaterials (amino acid). More specifically, it is possible to synthesizecereulide using L-valine, D-leucine and D-alanine, and synthesize aderivative by adding L-glutamic acid and 6-aminohexanoic acid to thesematerials.

Hereinbelow, the method of the present invention will be described indetail by each synthetic stage of the fragment. As a protective groupused in the production, for example, protective groups described in“Protective Groups in Organic Synthesis (1999)” from WILEY-Intersciencepublication are properly used.

First, as the amino acid required for synthesis, L-valine with anyprotective group attached thereto can be used in the synthesis, and acommercially available product with a protective group attached theretocan also be used as it is. Examples of the commercially availableproduct include Boc-L-valine protected with a tert-butoxycarbonyl groupmanufactured by Tokyo Chemical Industry Co., Ltd.

Next, D-leucine acid and L-valine acid can be respectively synthesizedfrom D-leucine and L-valine. Hydroxy acids such as D-leucine acid andL-valine acid can be used in any method as long as it is a method thatcan convert an amino group of D-leucine acid and L-valine acid into ahydroxyl group.

A representative example is a method of reacting D-leucine or L-valinewith a nitrite. It is preferred that D-leucine or L-valine be dissolvedin a strong acid such as sulfuric acid or hydrochloric acid, prior tothis reaction, and used.

Furthermore, it is also possible to attach a protective group to thehydroxy acid obtained as described above, in preparation for the nextreaction. As the protective group, a benzyl group and the like arepreferably used.

Similarly, a protective group can also be added to D-alanine to preparefor the next reaction.

Next, a didepsipeptide is synthesized using the amino acid and hydroxyacid obtained as described above.

A didepsipeptide may be synthesized under any condition as long as it isa condition that can achieve esterification by a dehydration reaction.Particularly preferred is a reaction carried out by adding a strong basesuch as dimethylaminopyridine and dicyclohexylcarbodiimide to a solventsuch as a dichloromethane solution, but the synthesis condition is notlimited thereto.

It is preferred to bind L-valine to D-leucine acid, then remove a groupprotecting the carboxyl group and use it in the subsequenttetradepsipeptide synthesis. Meanwhile, it is possible to bind D-alanineto D-valine acid, then remove the group protecting the amino group anduse it in the subsequent tetradepsipeptide synthesis.

Next, the obtained didepsipeptides are bound to synthesize atetradepsipeptide. At the time, the synthesis can be carried out underany condition as long as it is a condition that can form an amide bond.Preferably, a method of acting various condensing agents under neutralconditions can be adopted. A tetradepsipeptide serves as a repeatingunit for cereulide synthesis. For the ease of carrying out the nextsynthesis, it is also desirable to prepare two types of fragment A andfragment B to which the protective groups are each added to thedifferent portions of the terminals.

The protected repeating structure units that are each a key intermediateof cereulide synthesis, fragment A (12) and fragment B (13), weresynthesized in the above pathway.

Furthermore, the fragments obtained as described above are linked by anamide bond, thereby synthesizing an octadepsipeptide and furthersynthesizing a dodecadepsipeptide.

It is preferred that the linear dodecadepsipeptide obtained as describedabove be cyclized while preventing an intermolecular reaction, tosynthesize cereulide.

In more detail, it is possible to efficiently progress an intramolecularcyclization reaction by optimizing various conditions such as acondensing agent, a solvent and the temperature.

The condensing agent is not limited, but examples includeorganophosphorous compounds such as diphenylphosphoryl azide (DPPA),diethylphosphoryl cyanidate and azidotris(dimethylamino)phosphoniumhexafluorophosphate, quinoline-based peptide condensing agents such asN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and1-isobutyl-2-isobutyl-1,2-dihydroxyquinoline, uronium-based condensingagents such as O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) andO-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU), carbodiimides such as diisopropylcarbodiimide (DIC),dicyclohexylcarbodiimide (DCC) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, and phosphonium-basedcondensing agents such as(benzotriazole-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate, (benzotriazole-1-yloxy)trispyrrolidinophosphoniumhexafluorophosphate and bromotripyrrolizinophosphoniumhexafluorophosphate. Furthermore, these condensing agents can be usedtogether with an additive such as 1-hydroxy-7-azabenzotriazole (HOAt) or1-hydroxybenzotriazole (HOBt).

Examples of the solvent used for the reaction include methylenechloride, dimethylformamide and the like.

In the cyclization reaction, an intermolecular bond may be formed in anormal state, and thus it is preferred to perform the cyclizationreaction by a high dilution method.

In the present cyclization reaction, the intramolecular reactionprecedes an intermolecular reaction, and thus it is possible to increasethe yield of isolation of a subject matter. More specifically, it ispreferred to suppress the production of a by-product, and precede anintramolecular cyclization reaction.

The present invention further has high applicability in the synthesis ofa cereulide derivative (derivative). More specifically, the constituentamino acid or hydroxy acid in the repeating structure unit isappropriately changed, whereby simple synthesis of the derivative ismade possible.

For example, novel derivatives such as E-cereulide in which L-valine isreplaced by L-glutamic acid, and EAHA-cereulide obtained by introducingaminohexanoic acid into E-cereulide can be synthesized.

These new compounds are expected to be a useful tool molecule forelucidating physiological activity mechanisms of cereulide with manyunclear parts, including preparation of an anticereulide antibody.

More specifically, E-cereulide is a derivative in which one L-valine incereulide is replaced by L-glutamic acid, and EAHA-cereulide is one inwhich an omega-carboxyl group of E-cereulide is modified withaminohexanoic acid. In these two derivatives, a carboxyl group rich inreactivity is arranged in the external of a 36-membered ring via acarbon chain, and thus, further chemical modification is possible, andthese two derivatives can be used as a hapten molecule or labeledmolecule for preparing an anticereulide antibody.

Synthesis of such a cereulide derivative can be carried out by preparingone of three tetradepsipeptides L-Val-D-O-Leu-D-Ala-L-O-Val, that is arepeating unit of cereulide, of which L-valine is replaced withL-glutamic acid {R═(CH₂)₂COOH} or L-glutamic acid+aminohexanoic acid{R═(CH₂)₅COOH}.

That is, it is possible to carry out the synthesis specifically by thefollowing method. For example, the cereulide derivative can besynthesized using a commercially available protective L-glutamic acidderivative or the like as a starting material.

A didepsipeptide may be synthesized under any condition as long as it isa condition that can achieve esterification by a dehydration reaction.Particularly preferred is a reaction carried out by adding a strong basesuch as dimethylaminopyridine and dicyclohexylcarbodiimide to a solventsuch as a dichloromethane solution, but it is not limited thereto.

It is preferred to thereafter remove the group protecting the carboxylgroup and use it in the subsequent tetradepsipeptide synthesis.Meanwhile, it is possible to bind D-alanine to D-valine acid, thenremove the group protecting the amino group and use it in the subsequenttetradepsipeptide synthesis.

The obtained didepsipeptides are bound to synthesize atetradepsipeptide. The synthesis can be carried out under any conditionas long as it is a condition that can form an amide bond. Preferably, amethod of acting various condensing agents under neutral conditions canbe adopted. For the ease of carrying out the next synthesis, it isdesirable to prepare two types of fragment C and fragment D.

Furthermore, the fragments obtained as described above are linked by anamide bond, thereby synthesizing an octadepsipeptide and furthersynthesizing a dodecadepsipeptide. An example thereof is shown below,but is not limited thereto, and a method of linking tetradepsipeptides,the position of the protective group and the like can be appropriatelychanged.

Here, when synthesizing a depsipeptide, 34a, b having L-glutamic acid(Series a) or L-glutamic acid+aminohexanoic acid (Series b) may be boundso as to be sandwiched between fragment A and fragment B, and further,the amino group terminal of fragment 5 can also be bound to the carboxylgroup terminal of 34a, b, by appropriately changing the position of theprotective group.

It is preferred that the linear dodecadepsipeptide obtained as describedabove be cyclized while preventing an intermolecular reaction, tosynthesize a cereulide derivative:

wherein R represents (CH₂)₂COOH or (CH₂)₂CONH(CH₂)₅COOH.

The cereulide derivative as described above can be easily modified, andis useful for the production of an antigen for the antibody production.Furthermore, it is also possible to label the cereulide derivative, inorder to make a tool for elucidating physiological functions ofcereulide.

In order to make an antigen for the antibody production, it is preferredto bind a polymer such as bovine serum albumin (BSA) or keyhole lympethemocyanin (KLH) to the carboxyl group part of the cereulide derivative.

A labeled cereulide to be a tool for elucidating physiological functionscan be achieved, for example, by covalently binding a labeled substancesuch as biotin to the carboxyl group part of the cereulide derivative.

As a tool for revealing the physiological activity of cereulide at amolecular level, a photolabeled form was synthesized by introducingbiotin into the side chain of E-cereulide.

In each step of the synthesis as described above, the substance isproperly subjected to a neutralization step and a purification step, andthen may be subjected to the next step. Each product in each of thesteps as described above may be isolated and purified, or may bedirectly subjected to the next step. Examples of the isolation andpurification means include washing, extraction, a recrystallizationmethod, various chromatographic techniques and the like. Each product ineach step can be subjected to any one of these isolation andpurification means or any proper combination of two or more kinds ofthese isolation and purification means.

The cereulide and a derivative thereof obtained in the method of thepresent invention and a derivative thereof are obtained in a high yieldand have high purity, and thus are highly useful as a cereulidespecimen. In the present invention, it is also possible to provide acereulide detection kit containing a cereulide specimen. The cereulidedetection kit as described above can be provided with a solvent todissolve cereulide, a culture medium and the like. Furthermore,cereulide can be widely used for applications such as a potassium ionselective electrode utilizing cereulide's properties of incorporatingpotassium ion, and an anticancer drug utilizing an apoptosis action tocancer cells. Furthermore, it is also possible to provide more specificcancer cell selectivity by a combination with a drug delivery system orthe like.

The cereulide derivative of the present invention can be used as it isor in the form of a pharmaceutically acceptable salt, or in the form ofmixture of the cereulide derivative with a pharmaceutically acceptablecarrier as a preparation known to a person killed in the art.

Examples of the pharmaceutically acceptable salt include salts of aninorganic base, salts of an organic base, salts of an inorganic acid,salts of an organic acid, salts of a basic or acidic amino acid and thelike. Preferred examples of the salt of an inorganic base include alkalimetal salts such as sodium salts and potassium salts; alkaline earthmetal salts such as calcium salts and magnesium salts; and aluminumsalts, ammonium salts and the like. Preferred examples of the salt of anorganic base include salts of trimethylamine, triethylamine, pyridine,picoline, ethanolamine, diethanolamine, triethanolamine,dicyclohexylamine, N,N′-dibenzylethylenediamine or the like. Preferredexamples of the salt of an inorganic acid include salts of hydrochloricacid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid orthe like. Preferred examples of the salt of an organic acid includesalts of formic acid, acetic acid, trifluoroacetic acid, fumaric acid,oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid,malic acid, methanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid or the like. Preferred examples of the salt of abasic amino acid include salts of arginine, lysine, ornithine or thelike, and preferred examples of the salt of an acidic amino acid includesalts of aspartic acid, glutamic acid or the like.

Hereinbelow, specific examples of the production of cereulide andderivatives thereof of the present invention are described withreference to embodiments of examples, but the present invention is notlimited thereto.

EXAMPLES

In the following examples, analysis and separation and purification ofthe compound were carried out using the following models and reagents.

NMR spectrum: JEOL Ltd. JMTC-400/54/SS 400 MHz (manufactured by JEOLLtd.). Unless otherwise stated, a liquid sample was measured as a NaClfilm, a solid sample was measured as a KBr pellet, and the absorptionwavelength is expressed as cm⁻¹ in the sentence.) Also, the chemicalshift was expressed as a δ value.

Melting Point: measured using BUCHI Melting point B-545 (all meltingpoints not corrected).

IR: measured using JASCO FT/IR-460 plus. (Unless otherwise stated, aliquid sample was measured as a NaCl film, a solid sample was measuredas a KBr pellet, and the absorption wavelength is expressed as cm⁻¹ inthe sentence.)

Mass analysis: measured using MALDI-TOF-MS manufactured by BRUKERDALTONICS.

Thin-layer chromatography (TLC): developed using MACHEREY-NAGELDC-Fertigplatten SIL G-25 UV254 plate and using the following solventsystems. In the text, the solvent systems are expressed as the followingsymbols.

(A) n-hexane:ethyl acetate=3:1

(B) chloroform:methanol=9:1

(C) chloroform:methanol:acetic acid=85:15:3

(D) chloroform:methanol:triethylamine=90:10:3

(E) toluene:ethyl acetate=1:1

(F) n-hexane:ethyl acetate=1:1

Here, Examples 1 to 5 show the synthesis of the cereulide of the presentinvention, and Examples 6 to 10 show the synthesis of the cereulidederivative of the present invention.

Example 1

Synthesis of Boc Amino Acids

(1) Boc-L-valine (1)

A commercial (manufactured by Tokyo Chemical Industry Co., Ltd.) productwas used without purification.

mp; 78° C.

[a]_(D)−6.5° (c 1.0, AcOH)

(2) Boc-D-alanine (2)

Under ice cooling, D-alanine (11, 8.0 g, 89.8 mmol) was dissolved in anaqueous solution (150 mL) of sodium carbonate (19.1 g, 179.6 mmol), anda THF (20 mL) solution of di-tert-butyl dicarbonate (Boc₂O, 21.6 g, 98.8mmol) was added dropwise thereto. After completion of the dropwiseaddition, the reaction liquid was returned to room temperature, andcontinuously stirred overnight. The reaction liquid was transferred to aseparating funnel, and excess Boc₂O was removed by diethyl etherextraction. Citric acid was added to the aqueous layer to adjust the pHto 3, and then the mixture was extracted three times with ethyl acetate.The combined organic layer was washed once with a saturated salinesolution, and the organic layer was dried over anhydrous Na₂SO₄, andconcentrated under reduced pressure, and then the concentrated residuewas recrystallized from ethyl acetate-hexane to obtain a colorlesscrystal 2 (15.51 g, 91.3%).

TLC; Rf=0.48 (C)

mp; 83.5° C.

¹H-NMR (CDCl₃); 1.40-1.45 (12H, m), 4.24-4.38 (1H, m, CHCH₃), 4.98-5.10(1H, br s, NH)

[a]_(D)+25.1 (AcOH, c 2.02, 26.6° C.)

IR; 3378, 2995, 2639, 2569, 1736, 1161

Synthesis of Hydroxy Acids (3, 5) and Benzyl Esters Thereof (4, 6)

(3) D-O-leucine {(2R)-2-hydroxy-4-methylpentanoic acid} (3)¹⁾

Under ice cooling, D-leucine (13.11 g, 100 mmol) was dissolved in 1 Nsulfuric acid (150 mL), and an aqueous solution (50 mL) of sodiumnitrite (10.35 g, 150 mmol) was added dropwise thereto from a droppingfunnel. The reaction liquid was stirred for 3 hours under ice cooling,then further stirred for 6 hours under room temperature, and extractedthree times with ethyl acetate. The combined organic layer was washedonce with a saturated saline solution, dried over anhydrous sodiumsulfate, and concentrated under reduced pressure. The concentratedresidue was recrystallized from ethyl acetate-hexane to obtain acolorless crystal D-O-leucine 3 (9.60 g, 72.7%).

TLC; Rf=0.33 (C)

mp; 80.1° C. (lit.¹⁾ 78° C.)

¹H-NMR (DMSO); 0.36 (3H, d, J=6.34 Hz, CH(CH₃ )₂), 0.38 (3H, d, J=6.34Hz, CH(CH₃ )₂), 0.85-0.98 (2H, m, CH₂ ), 1.17-1.32 (1H, m, CH(CH₃)₂),3.43 (1H, dd J₁=8.78, J₂=4.88, CHCH₂)

¹³C-NMR (DMSO); 21.55, 23.24, 23.97, 42.98, 68.22, 176.41

[a]_(D)+11.7° (MeOH, c 1.03, 22.5° C.) {lit.¹⁾+11.8° (MeOH, c 1.03)}

IR; 3424, 2911, 1713

(4) L-O-valine {(2S)-2-hydroxy-3-methylbutanoic acid} (5)

L-O-valine (5) was prepared in the same manner as in the synthesis of 3,using commercially available L-valine as a raw material. Morespecifically, under ice cooling, an aqueous solution (50 mL) of sodiumnitrite (10.35 g, 150 mmol) was added dropwise to a 1 N sulfuric acid(150 mL) solution of L-valine (11.71 g, 100 mmol), and after completionof the dropwise addition, the mixture was stirred for 3 hours under icecooling, and subsequently for 6 hours under room temperature. Thereaction liquid was extracted three times with ethyl acetate, thecombined organic layer was washed once with a saturated saline solution,a trace amount of contained sulfuric acid was removed, and the organiclayer was dried over anhydrous sodium sulfate, and concentrated underreduced pressure. The concentrated residue was recrystallized from ethylacetate-hexane to obtain 5 (7.07 g, 59.9%) as a colorless crystal.

TLC; Rf=0.37 (C)

mp; 64.1° C. (lit.²⁾ 66-68° C.)

¹H-NMR (DMSO); 0.80 (3H, d, J=3.17 Hz, CH(CH₃ )₂), 0.87(3H, d, J=3.17Hz, CH(CH₃ )₂), 1.85-1.95 (1H, m, CH(CH₃)₂), 3.72 (1H, d, J=4.63 Hz,CHCH(CH₃)₂), 5.02 (1H, br, COOH)

¹³C-NMR (DMSO); 16.89, 19.05, 31.55, 74.64, 175.51

[a]_(D)+16.8° (CHCl₃, c 1.01, 23.5° C.) {lit.²⁾; +20° (CHCl₃, c 4)}

IR; 3433, 2970, 2186, 1714

(5) D-O-leucine benzyl ester {benzyl (2R)-2-hydroxy-4-methylpentanoate}(4)

A mixture of D-O-leucine (3, 3.36 g, 25.4 mmol), benzyl alcohol (7.9 mL,76.0 mmol), p-toluenesulfonic acid monohydrate (50 mg, 0.26 mmol) andtoluene (100 mL) was heated under reflux in a recovery flask equippedwith a Dean-Stark apparatus for 7 hours to remove the generated water byazeotropic separation. After confirming disappearance of the rawmaterial by TLC, the reaction liquid was returned to room temperature,and washed with a saturated aqueous solution of sodium hydrogencarbonate, followed by a saturated saline solution. The organic layerwas dried over anhydrous sodium sulfate, and concentrated under reducedpressure with an evaporator. The benzyl alcohol contained in theconcentrated residue was distilled away under reduced pressure (8 mHg<),using a glass tube oven (bulb to bulb distillation) apparatus. Thisresidue was purified by silica gel column chromatography (hexane:ethylacetate=90:10) to obtain a pale yellow oily matter 4 (4.95 g, 80.8%).

TLC; Rf=0.38 (A)

¹H-NMR (CDCl₃); 0.91 (3H, d, J=4.39 Hz, CH(CH₃ )₂), 0.94 (3H, d, J=4.39Hz, CH(CH₃ )₂), 1.56-1.62 (2H, m, d, CH₂ ), 2.60-2.65 (1H, m, OH),4.18-4.26 (1H, m, CHCH₂), 5.19 (2H, ABq, CH₂ Ph), 7.30-7.40 (5H, m, ArH)

¹³C-NMR (CDCl₃); 21.52, 23.21, 24.39, 67.25, 69.15, 128.30, 128.30,128.52, 128.64, 135.21, 175.71)

[a]_(D)+15.4° (CHCl₃, c 1.02, 26.8° C.)

IR; 3475, 2956, 2872, 1736, 1607, 1497, 1140

(6) L-O-valine benzyl ester {benzyl (2S)-2-hydroxy-3-methylbutanoate}(6)

A benzyl ester (6) was synthesized in the same manner as in 4 describedabove.

A mixture of L-O-valine (5, 3.0 g, 25.4 mmol), benzyl alcohol (7.9 mL,76.0 mmol), p-toluenesulfonic acid monohydrate (50 mg, 0.26 mmol) andtoluene (100 mL) was heated under reflux in a reaction vessel equippedwith a Dean-Stark apparatus for 7 hours, and subjected to the sametreatment and purification to obtain a pale yellow oily matter 6 (4.57g, 86.4%).

TLC; Rf=0.44 (A)

¹H-NMR (CDCl₃); 0.83 (3H, d, J=6.09 Hz, CH(CH₃ )₂), 1.01 (3H, d, J=6.09Hz, CH(CH₃ )₂), 2.02-2.14 (1H, m, CH(CH₃)₂), 2.69 (1H, d, J=6.09, OH),4.06-4.11 (1H, m, CHCH), 5.22 (2H, ABq, CH₂ Ph), 7.31-7.60 (5H, m, ArH)

¹³C-NMR (CDCl₃); 15.82, 18.77, 32.14, 67.29, 74.98, 128.41, 128.56,128.64, 135.16, 174.82

[a]_(D)−9.60° (CHCl₃, c 2.19, 26.0° C.)

IR; 3505, 3065, 3034, 2965, 2934, 2876, 1733, 1497, 1460, 1137

Example 2

Synthesis of Didepsipeptides (7, 8)

(7) Boc-L-Val-D-O-Leu-OBn (7)

Under ice cooling, dimethylaminopyridine (DMAP, 0.55 g, 4.50 mmol) wasadded to a dichloromethane (80 mL) solution of D-O-leucine benzyl ester(4, 5.0 g, 22.5 mmol) and commercially available Boc-L-valine (1, 5.38g, 24.8 mmol), and then N,N′-dicyclohexyl carbodiimide (DCC, 5.86 g,28.4 mmol) was added thereto. This reaction liquid was stirred under icecooling overnight, then the by-produced DCurea was removed by suctionfiltration, and the filtrate was concentrated under reduced pressure.The concentrated residue was dissolved in ethyl acetate and washed witha saturated aqueous solution of sodium hydrogen carbonate, followed by asaturated saline solution, and the organic layer was dried overanhydrous sodium sulfate, and concentrated under reduced pressure. Thisconcentrated residue was purified by silica gel column chromatography(hexane/ethyl acetate) to obtain a colorless solid 7 (9.30 g, 98%).

TLC; Rf=0.56 (A)

¹H-NMR (CDCl₃); 0.78-0.92 (12H, m, CH(CH₃ )₂), 1,37 (9H, s, tert-Bu),1.50-1.82 (3H, m), 2.02-2.20 (1H, m, CH(CH₃)₂), 4.18-4.32 (1H, m,CHCH₂CH(CH₃)₂), 4.91 (1H, d, J=8.78 Hz, NH), 5.04 (1H, dd, J₁=10.0 Hz,J₂=3.66 Hz), 5.10 (2H, ABq, CH₂ Ph), 7.28-7.36 (5H, m, ArH)

¹³C-NMR (CDCl₃); 17.31, 19.04, 21.27, 23.04, 24.49, 28.30, 31.23, 39.62,58.62, 67.04, 71.59, 79.72, 128.22, 128.41, 128.59, 135.22, 155.49,170.12, 171.78

[a]_(D)+15.58° (CHCl₃, c 0.40, 16.6° C.)

IR; 3384, 2964, 2875, 1746, 1717, 1501, 1462, 1177

(8) Boc-L-Val-D-O-Leu-OH (8) (Removal of Benzyl Group by Hydrogenolysis)

A flask for hydrogenation was charged with a mixture ofBoc-L-Val-D-O-Leu-OBn (7, 2.70 g, 6.40 mmol), methanol (30 mL) and 10%palladium carbon (135 mg), and the mixture was stirred under a hydrogenstream (3 atmospheres) at room temperature for 3 hours. After confirmingthe progress of the reaction by TLC, the catalyst was removed byfiltration, and the filtrate was concentrated under reduced pressure toobtain a colorless solid 8 (2.14 g, quant). This solid was used for thenext reaction without further purification.

TLC; Rf=0.55 (C)

mp; 95.8° C.

¹H-NMR (CDCl₃); 0.86-1.06 (12H, m, CH(CH₃ )₂), 1.44 (9H, s, tert-Bu),1.72-1.80 (2H, m, CH₂ CH(CH₃)₂), 1.80-1.86 (1H, m, CHCH(CH₃)₂),2.14-2.28 (1H, m, CH₂CH(CH₃)₂), 4.22-4.33 (1H, m, CHCH₂), 5.00-5.06 (1H,m, NH), 5.06-5.14 (1H, m, CHCH(CH₃)₂)

¹³C-NMR (CDCl₃); 17.49, 19.06, 21.17, 23.06, 24.54, 28.28, 30.93, 39.62,52.49, 58.86, 69.99, 71.19, 80.19, 113.79, 121.42, 155.84, 174.30

IR; 3516, 3396, 3093, 2961, 2606, 1726, 1520, 1463, 1409, 1178

(9) Boc-D-Ala-L-O-Val-OBn (9)⁴⁾

A didepsipeptide (9) was synthesized in the same manner as in 7. Morespecifically, Boc-D-alanine (2, 4.46 g, 23.6 mmol), L-O-valine benzylester (6, 4.10 g, 19.7 mmol) and dichloromethane (50 mL) were stirredunder ice cooling. DMAP (0.72 g, 5.90 mmol) was added thereto, then DCC(5.07 g, 24.59 mmol) was added thereto, and the mixture was stirredunder ice cooling overnight. The by-produced DCurea was removed bysuction filtration, the filtrate was concentrated under reducedpressure, and then the concentrated residue was dissolved in ethylacetate. This ethyl acetate solution was washed with a saturated aqueoussolution of sodium hydrogen carbonate, followed by a saturated salinesolution, and the organic layer was dried over anhydrous sodium sulfate,and concentrated under reduced pressure. The concentrated residue waspurified by silica gel column chromatography (hexane/ethyl acetate) toobtain a colorless solid 9 (7.66 g, quant).

TLC; Rf=0.56 (A)

mp; 63.4° C.

¹H-NMR (CDCl₃); 0.94 (3H, d, J=6.83 Hz, CH(CH₃ )₂), 0.98 (3H, d, J=6.83Hz, CH(CH₃ )₂), 1.41 (3H, d, J=7.07 Hz, CHCH₃ ), 1.45 (9H, s, tert-Bu),2.22-2.34 (1H, m, CH(CH₃)₂), 4.43-4.50 (1H, m, CHCH₃), 4.93 (1H, d,J=4.39, CHCHCH₃), 5.03 (1H, br, NH), 5.18 (2H, ABq, CH₂ Ph), 7.28-7.40(5H, m, ArH)

¹³C-NMR (CDCl₃); 17.01, 18.59, 18.70, 28.31, 30.12, 49.33, 66.97, 79.76,128.32, 128.43, 135.22, 154.91, 169.06, 172.74

IR; 3396, 2976, 1741, 1688, 1509, 1459, 1161

[a]_(D)−10.70° (CHCl₃, c 1.02, 27.0° C.)

(10) H₂N-D-Ala-L-O-Val-OBn TFA (10) (Removal of Boc Group)

Under ice cooling, trifluoroacetic acid (TFA, 7.5 mL) was added to adichloromethane (7.5 mL) solution of Boc-L-Val-D-O-Leu-OBn (9) (2.43 g,6.40 mmol). This reaction liquid was stirred for 30 minutes under icecooling, and subsequently for 30 minutes under room temperature. Afterconfirming by TLC that the Boc group was removed, the reaction liquidwas concentrated under reduced pressure to obtain a pale yellow oilymatter 10 (2.41 g, quant) as a TFA salt. This oily matter was used forthe next reaction without further purification.

TLC; Rf=0.51 (B)

¹H-NMR (CDCl₃); 0.82 (3H, d, J=6.83 Hz, CH(CH₃ )₂), 0.87 (3H, d, J=6.83Hz, CH(CH₃ )₂), 1.54 (3H, d, J=7.32 Hz, CHCH₃ ), 2.12 (1H, m, CH(CH₃)₂),4.12 (1H, q, J=7.32 Hz, CHCH₃), 4.91 (1H, d, J=4.15, CHCH(CH₃)₂), 5.06(2H, ABq, CH₂ Ph), 7.20-7.30 (5H, m, ArH)

¹³C-NMR (CDCl₃); 15.54, 16.77, 18.40, 30.05, 49.01, 67.39, 78.35,128.45, 128.57, 128.60, 134.96, 168.69, 169.70

IR; 3420, 3035, 2970, 1742, 1674, 1527, 1461, 1141

Example 3 (11) Synthesis of Tetradepsipeptide, Fragment A and Fragment B(11) Boc-L-Val-D-O-Leu-D-Ala-L-O-Val-OBn (11)

Under ice cooling, N,N-diisopropylethylamine (DIPEA, 1.12 mL, 6.43 mmol)was added to an acetonitrile (10 mL) solution of didepsipeptideH₂N-D-Ala-L-O-Val-OBn TFA salt (10, 2.41 g, 6.40 mmol) to neutralize thesolution, then an acetonitrile solution (5 mL) of Boc-L-Val-D-O-Leu-OH(8, 2.14 g, 6.40 mmol) was added, and subsequently1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride (1.35g, 7.04 mmol) was added. The ice cooling bath was removed, and thereaction liquid was stirred under room temperature overnight, and thenthe solvent was distilled away under reduced pressure. This residue wasdissolved in ethyl acetate, and the organic layer was washed with a 10%aqueous solution of citric acid, a saturated aqueous solution of sodiumhydrogen carbonate, followed by a saturated saline solution, dried overanhydrous sodium sulfate, and concentrated under reduced pressure. Thisresidue was purified by silica gel column chromatography (hexane/ethylacetate) to obtain a colorless oily matter 11 (3.56 g, 94%).

TLC; Rf=0.33 (A)

¹H-NMR (CDCl₃); 0.84-1.04 (18H, m, CH(CH₃ )₂), 1.41 (9H, s, tert-Bu),1.64-1.84 (3H, m), 2.06-2.15 (1H, m, CHCH(CH₃)₂), 2.18-2.30 (1H, m, CH₂CH(CH₃)₂), 4.06-4.17 (1H, m, CHCH₃), 4.52-4.63 (1H, m, CHCH₂CH(CH₃)₂),4.86-4.94 (1H, m, CHCH(CH₃)₂), 5.00-5.05 (1H, m, NH), 5.08-5.27 (2H, m,CH₂ Ph), 5.26-5.34 (1H, m, CHCH(CH₃)₂), 7.09-7.14 (1H, br, NH),7.28-7.38 (5H, m, ArH)

¹³C-NMR (CDCl₃); 16.97, 17.24, 18.06, 18.68, 19.17, 21.38, 23.20, 24.33,28.25, 30.10, 30.44, 34.54, 40.51, 48.53, 59.53, 63.52, 66.96, 72.66,80.24, 100.55, 128.36, 128.40, 128.55, 135.27, 155.89, 169.11, 169.79,170.43, 171.77

[a]_(D)+16.03 (CHCl₃, c 0.62, 23.5° C.)

IR; 3344, 2968, 2878, 1756, 1681, 1530, 1462, 1056

(12) H₂N-L-Val-D-O-Leu-D-Ala-L-O-Val-OBn TFA (12) (Fragment A)

The protected repeating structure unit (11, 2.43 g, 6.40 mmol) wasdissolved in dichloromethane (7.5 mL), TFA (7.5 mL) was added under icecooling, the mixture was stirred for 30 minutes, and then stirred atroom temperature for 30 minutes. After confirming completion of thereaction by TLC, the reaction liquid was concentrated under reducedpressure. A small amount of toluene was added to this residue, themixture was concentrated under reduced pressure, and the remaining TFAwas removed by co-evaporation with toluene. This operation was repeatedthree times to obtain a pale yellow oily matter 12 (2.42 g, quant). Thisoily matter was used for the next reaction without further purification.

TLC; Rf=0.59 (C)

¹H-NMR (CDCl₃); 0.88-1.16 (18H, m, CH(CH₃ )₂), 1.52 (3H, d, J=7.32 Hz,CHCH₃ ), 1.70-1.85 (6H, m), 2.18-2.30 (1H, m, CH₂ CH(CH₃)₂), 2.38-2.51(1H, m, CH₂ CH(CH₃)₂), 4.09-4.15 (1H, m, CHCH₃), 4.58-4.67 (1H, m,CHCH₂CH(CH₃)₂), 4.86-4.94 (1H, m, CHCH(CH₃)₂), 5.00-5.05 (1H, m, NH),5.08-5.27 (2H, m, CH₂ Ph), 5.26-5.34 (1H, m, CHCH(CH₃)₂), 7.09-7.14 (1H,br, NH), 7.28-7.38 (5H, m, ArH)

¹³C-NMR (CDCl₃); 17.04, 17.08, 18.22, 18.35, 18.61, 21.52, 22.98, 24.30,29.94, 30.14, 40.74, 48.47, 58.90, 67.02, 74.32, 128.32, 128.40, 128.54,135.16, 168.33, 169.25, 169.40, 172.25

IR; 3221, 2964, 1748, 1675, 1531, 1461, 1156

(13) Boc-L-Val-D-O-Leu-D-Ala-L-O-Val-OH (13) (Fragment B)

A flask for medium-pressure hydrogenation was charged with a mixture ofa methanol solution (20 mL) of the protected repeating structure unit(11, 2.70 g, 6.40 mmol) and 10% palladium carbon (135 mg), and themixture was stirred under a hydrogen stream (3 atmospheres) at roomtemperature for 4 hours. After confirming the progress of the reactionby TLC, the solvent was distilled away under reduced pressure to obtaina colorless oily matter 13 (2.12 g, quant). This oily matter was usedfor the next reaction without purification.

TLC; Rf=0.18 (C)

¹H-NMR (CDCl₃); 0.87-1.04 (18H, m, CH(CH₃ )₂), 1.42-1.50 (12H, m),1.60-1.81 (3H, m), 2.09-2.18 (1H, m, CHCH(CH₃)₂), 2.25-2.35 (1H, m, CH₂CH(CH₃)₂), 4.13-4.23 (1H, m, CHCH₃), 4.52-4.62 (1H, m, CHCH₂CH(CH₃)₂),5.04-5.12 (2H, m), 5.32-5.38 (1H, m, CHCH(CH₃)₂), 7.34-7.39 (1H, d,J=7.56 Hz, NH)

¹³C-NMR (CDCl₃); 16.78, 16.83, 17.87, 18.78, 19.12, 21.20, 23.20, 24.30,28.21, 29.93, 30.39, 40.60, 48.28, 59.40, 72.80, 77.21, 80.64, 156.33,170.45, 171.82, 171.94

IR; 3341, 2968, 2882, 1748, 1688, 1531, 1464, 1371, 1249, 1162, 1057,1016, 757

Example 4

Synthesis of Octadepsipeptide and Dodecadepsipeptide

(14) Boc-(L-Val-D-O-Leu-D-Ala-L-O-Val)₂-OBn (14)

Under ice cooling, N,N-diisopropylethylamine (DIPEA, 1.03 mL, 5.91 mmol)was added to an acetonitrile (15 mL) solution of fragment A TFA salt(12, 1.75 g, 3.0 mmol), and an acetonitrile (5 mL) solution of fragmentB (13, 1.49 g, 3.0 mmol), O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU, 1.41 g, 3.71 mmol) and 1-hydroxybenzotriazole(HOBt, 0.40 g, 3.0 mmol) were subsequently added. The reaction liquidwas returned to room temperature and stirred overnight, and then thesolvent was distilled away under reduced pressure. This residue wasdissolved in ethyl acetate, and the ethyl acetate layer was sequentiallywashed with a 10% aqueous solution of citric acid, saturated sodiumhydrogen carbonate and a saturated saline solution, and dried overanhydrous sodium sulfate. The resulting substance was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (hexane/ethyl acetate) to obtain a colorless solid 14(2.90 g, quant).

TLC; Rf=0.14 (A)

mp; 57 to 59° C.

¹H-NMR (CDCl₃); 0.84-1.07 (36H, m), 1.43 (9H, s), 1.47 (3H, d, J=7.56Hz), 1.49 (3H, d, J=7.32 Hz), 1.65-1.84 (6H, m), 1.97-2.05 (1H, m),2.29-2.37 (1H, m), 2.37-2.38 (1H, m), 3.88 (3H, t, J=6.59 Hz), 4.08-4.13(1H, m), 4.38 (1H, t, J=7.81 Hz), 4.58-4.63 (1H, m), 4.85 (1H, d, J=4.39Hz), 5.03 (1H, d, J=5.85 Hz), 5.09-5.20 (4H, m), 5.37 (1H, dd, J=3.17,10.0 Hz), 5.04-5.12 (2H, m), 7.30-7.37 (5H, m), 7.68 (1H, d, J=7.32 Hz),7.68 (1H, d, J=7.32 Hz), 7.68 (1H, d, J=6.10 Hz)

¹³C-NMR (CDCl₃); 14.17, 16.43, 16.97, 17.51, 18.64, 18.93, 18.98, 19.31,19.45, 20.80, 21.05, 21.10, 23.14, 23.36, 24.22, 24.34, 28.22, 29.82,30.04, 30.09, 40.35, 40.99, 48.25, 49.52, 58.74, 60.34, 60.39, 66.73,72.41, 72.73, 77.21, 78.68, 80.93, 128.24, 128.28, 128.49, 135.36,156.45, 169.12, 170.00, 170.42, 170.67, 171.65, 171.91, 172.44

IR; 3319, 2966, 2876, 1751, 1656, 1537, 1463, 1185

[a]_(D)+12.7° (CHCl₃, c 1.01, 26.5° C.)

(15) Boc-(L-Val-D-O-Leu-D-Ala-L-O-Val)₂-OH (15)

A flask for hydrogenation was charged with a mixture of the protectedoctadepsipeptide 14 (1.00 g, 1.02 mmol), methanol (30 mL) and 10%palladium carbon (50 mg), and the mixture was stirred under a hydrogenstream (3 atmospheres) at room temperature for 4 hours. After confirmingthe progress of the reaction by TLC, the palladium catalyst was removedby filtration and concentrated under reduced pressure to obtain acolorless oily matter 15 (0.88 g, 97%). This oily matter was used forthe next reaction without purification.

TLC; Rf=0.67 (C)

mp; 85 to 87° C.

¹H-NMR (CDCl₃); 0.80-1.06 (36H, m), 1.43 (9H, s), 1.47 (3H, d, J=7.07Hz), 1.52 (3H, d, J=7.07 Hz), 1.62-1.84 (6H, m), 1.96-2.08 (1H, m),2.19-2.38 (3H, m), 3.93 (1H, t, J=6.83 Hz), 4.12-4.22 (1H, m), 4.22-4.31(1H, m), 4.44-4.61 (1H, m), 4.94-5.02 (2H, m), 5.09 (1H, d, J=6.09 Hz),7.65 (1H, d, J=6.83 Hz), 7.74 (1H, d, J=5.85 Hz), 7.83 (1H, d, J=7.32Hz)

The spectrum is shown in FIG. 1.

¹³C-NMR (CDCl₃); 16.28, 16.65, 16.87, 18.74, 19.01, 19.21, 19.26, 21.04,23.22, 23.26, 24.35, 28.23, 29.53, 29.93, 30.18, 30.35, 40.41, 40.65,48.00, 49.37, 59.28, 60.12, 72.69, 77.21, 78.82, 80.84, 156.46, 170.62,170.71, 170.88, 171.12, 171.22, 171.53, 171.75, 172.33

IR; 3060, 2966, 2876, 1751, 1658, 1535, 1465, 1389, 1370, 1301, 1241,1155, 1058, 1010, 931, 877, 838, 782, 628

(16) Boc-L-Val-D-O-Leu-D-Ala-L-O-Val)₃-OBn (16)

Under ice cooling, an acetonitrile (5 mL) solution of fragment B (13)TFA salt (479 mg, 0.81 mmol) was charged, and neutralized by addingDIPEA (0.14 mL, 0.81 mmol), and then an acetonitrile (5 mL) solution ofoctadepsipeptide 15 (720 mg, 0.81 mmol) was added. Subsequently,O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 334 mg, 0.88 mmol) was added. The reactionliquid was returned to room temperature and stirred overnight, thenconcentrated under reduced pressure, and the residue was dissolved inethyl acetate. The ethyl acetate layer was sequentially washed with a10% aqueous solution of citric acid, a saturated aqueous solution ofsodium hydrogen carbonate and a saturated saline solution. The organiclayer was dried over anhydrous sodium sulfate, and then concentratedunder reduced pressure, and the residue was purified by silica gelcolumn chromatography (hexane/ethyl acetate=7:3) to obtain a colorlesssolid 16 (905 mg, 82%).

TLC; Rf=0.33 (F)

mp; 71 to 73° C.

¹H-NMR (CDCl₃); The spectrum is shown in FIG. 2.

¹³C-NMR (CDCl₃); 14.06, 16.27, 16.34, 16.41, 16.53, 17.09, 17.15, 17.68,18.57, 18.94, 18.98, 19.05, 19.17, 19.20, 19.31, 19.40, 20.69, 20.81,21.12, 23.03, 23.15, 23.31, 24.20, 24.35, 28.23, 29.74, 30.02, 30.11,30.15, 30.38, 40.53, 40.90, 41.18, 48.17, 49.36, 49.62, 58.27, 58.73,60.40, 66.62, 72.41, 72.74, 77.21, 78.88, 79.09, 80.91, 128.20, 128.45,135.41, 156.56, 169.22, 169.71, 169.96, 170.36, 170.51, 170.56, 170.81,171.90, 172.45

[a]_(D)+6.12° (CHCl₃, c 1.02, 25.9° C.)

IR; 3320, 2965, 2876, 1751, 1655, 1538, 1466, 1370, 1336, 1242, 1184,1153, 1058, 1006, 933, 876, 747, 697

(17) Boc-(L-Val-D-O-Leu-D-Ala-L-O-Val)₃-OH (17)

A reaction vessel for hydrogenation was charged with a mixture of theprotected dodecadepsipeptide 16 (133 mg, 97.7 μmol), methanol (5 mL) and10% palladium carbon (7 mg), and the mixture was stirred under ahydrogen stream (3 atmospheres) at room temperature for 3 hours. Afterconfirming the progress of the reaction by TLC, the solvent wasdistilled away under reduced pressure to obtain a colorless solid 17(120.2 mg, 97%). This was used for the next reaction withoutpurification.

TLC; Rf=0.33 (B)

mp; 97 to 99° C.

¹H-NMR (CDCl₃); The spectrum is shown in FIG. 3.

¹³C-NMR (CDCl₃); 16.33, 16.39, 16.48, 16.60, 16.78, 16.88, 18.78, 18.84,18.94, 19.03, 19.18, 19.24, 19.26, 19.31, 20.92, 21.09, 23.18, 23.34,24.32, 24.39, 28.28, 29.51, 29.86, 29.92, 30.21, 30.22, 40.52, 40.82,48.26, 49.35, 49.54, 58.82, 59.06, 60.30, 72.77, 72.82, 73.02, 77.23,77.58, 78.99, 79.04, 80.89, 156.55, 170.46, 170.65, 170.84, 170.97,171.21, 171.55, 172.11, 172.36, 172.53

IR; 3319, 3068, 2965, 2938, 2876, 1751, 1658, 1536, 1467, 1389, 1370,1335, 1308, 1249, 1185, 1154, 1128, 1109, 1058, 1008, 933, 877, 755

(18) H-(L-Val-D-O-Leu-D-Ala-L-O-Val)₃-OH (18) TFA Salt

Under ice cooling, TFA (2 mL) was added to a dichloromethane (2 mL)solution of 17 (181 mg, 142 μmol), and the mixture was continuouslystirred under ice cooling for 1 hour. After confirming the progress ofthe reaction by TLC, toluene was added to the reaction liquid, and themixture was concentrated under reduced pressure. In order to completelyremove TFA, toluene was added to the concentrated residue and TFA wasazeotropically distilled away. This operation was repeated three timesto obtain a colorless solid 18 (181 mg, quant). This solid was used forthe next cyclization reaction without purification.

TLC; Rf=0.45 (B)

mp; 118 to 121° C.

¹H-NMR (CDCl₃); The spectrum is shown in FIG. 4.

IR; 3310, 3060, 2965, 2882, 2360, 1749, 1659, 1542, 1466, 1389, 1208,1154, 1059, 1007

Example 5

Synthesis of Cereulide (Cyclization Reaction)

(19) Cereulide

In a reaction vessel replaced with argon, diphenylphosphoryl azide(DPPA) (9.8 μL, 45.5 μmol) was dissolved in anhydrousN,N-dimethylformamide (12 mL), and then N,N-diisopropylethylamine (15.7μL, 90 μmol) was added thereto to prepare a liquid A. Separately, aprecursor 18 (40 mg, 31.5 μmol) was dissolved in anhydrousN,N-dimethylformamide (8 mL) to prepare a liquid B. Under roomtemperature, the liquid B was added dropwise to the liquid A using amicrosyringe over 1 hour or more, and continuously stirred under roomtemperature for 10 days. The reaction liquid was concentrated underreduced pressure to distill N,N-dimethylformamide away, and the residuewas dissolved in ethyl acetate, and sequentially washed with a saturatedaqueous solution of sodium hydrogen carbonate, 1 N hydrochloric acid anda saturated saline solution. The organic layer was dried over anhydroussodium sulfate, and concentrated under reduced pressure, and the residuewas purified by silica gel column chromatography (hexane/ethylacetate=7:3) to obtain cereulide (5.2 mg, 14%) as a pale yellow solid.This cereulide was recrystallized from hot n-hexane to obtain purecereulide as a colorless crystal.

TLC; Rf=0.23 (F)

mp; 200° C. (lit.⁵⁾ 196-199° C.)

¹H-NMR (CDCl₃); 0.85-1.02 (m, 45H), 1.06 (d, J=6.59 Hz, 9H), 1.46 (d,J=7.07 Hz, 9H), 1.59-1.85 (m, 9H), 2.24-2.39 (m, 6H), 4.10 (dd, J=9.27,7.81 Hz, 3H), 4.37-4.42 (m, 3H), 5.01 (d, J=3.17 Hz, 3H), 5.32 (dd,J=7.81, 5.12 Hz, 3H), 7.81 (d, J=6.83 Hz, 6H)

The spectrum is shown in FIG. 5.

¹³C-NMR (CDCl₃); 15.75, 16.85, 18.55, 19.28, 21.20, 23.34, 24.37, 28.62,30.48, 40.54, 48.78, 9.45, 72.69, 78.62, 170.49, 171.07, 171.49, 171.87

[a]_(D)+10.5° C. (CHCl₃, c 0.78, 25° C.), {lit.⁵⁾+3.37° (CHCl₃, c 1.03)}

IR; 3303, 2963, 2875, 1744, 1656, 1539, 1467, 1246, 1190, 1149, 1057,1008

MALDI/TOFMS; 1175.576 [M+Na]^(|), 1191.561 [M+K]^(|)

Example 6

6-Aminohexanoic Acid (AHA) and L-Glutamic Acid Derivative

(1) 6-Aminohexanoic acid benzyl ester (AHA-OBn, 20) p-TosOH Salt

Commercially available 6-aminohexanoic acid (AHA, 19) (6.56g, 50.0mmol), benzyl alcohol (15.6 mL, 150 mmol), p-TosOH monohydrate (11.41 g,60.0 mmol) and toluene (150 mL) were charged in a recovery flaskequipped with a Dean-Stark apparatus, and the mixture was heated underreflux for 7 hours to remove the generated water by azeotropicseparation. After confirming disappearance of the raw material by TLC,the reaction liquid was returned to room temperature, hexane was added,and then the precipitated crystal was filtered by suction. The crystalwas well washed with cold ethyl acetate to obtain a colorless solid 20(18.71 g, 95.1%).

TLC; Rf=0.35 (D)

mp; 107.9° C.

¹H-NMR (CD₃OD); 1.29-1.44 (2H, m), 1.54-1.69 (4H, m), 2.33-3.34 (5H, m),2.80-2.90 (2H, m), 3.26-3.34 (2H, m), 5.11 (2H, s), 7.22 (2H, d,J=7.81), 7.28-7.37 (5H, m), 7.66-7.72 (2H, m)

¹³C-NMR (CD₃OD); 21.31, 25.37, 26.79, 28.17, 34.60, 40.50, 67.21,126.92, 129.92, 129.23, 129.26, 129.55, 129.85, 137.65, 141.75, 143.45,174.86

IR; 3050, 2945, 2634, 2042, 1730, 1622, 1476, 1248

(2) Troc-L-glutamic acid g-benzyl ester (22a)

Under ice cooling, a commercially available (manufactured by WATANABECHEMICAL INDUSTRIES, LTD.) L-glutamic acid g-benzyl ester 21 (3.00 g,12.6 mmol) was dissolved in an aqueous solution (60 mL) of sodiumhydrogen carbonate (3.19 g, 38.0 mmol), and a diethyl ether (10 mL)solution of 2,2,2-trichloroethyl chloroformate (Troc-Cl) (2.20 mL, 16.0mmol) was added dropwise thereto. After completion of the dropwiseaddition, the reaction liquid was returned to room temperature, andcontinuously stirred overnight. The reaction liquid was transferred to aseparating funnel, and excess Troc-Cl was removed by diethyl etherextraction. The aqueous layer was adjusted to pH 3 by adding citric acidand then extracted three times with ethyl acetate. The combined organiclayer was washed once with a saturated saline solution, dried overanhydrous sodium sulfate, and concentrated under reduced pressure, toobtain a colorless oily matter 22a (3.95 g, 75.7%) as the residue. Thisoily matter was used for the next reaction without purification.

TLC; Rf=0.56 (A)

¹H-NMR (CDCl₃); 2.07-2.19 (1H, m), 2.18-2.29 (1H, m), 2.31-2.43 (1H, m),2.46-2.65 (2H, m), 4.54-4.63 (1H, m), 4.66-4.78 (2H, m), 5.14 (2H, s),5.72 (1H, d, J=8.05 Hz), 7.30-7.40 (5H, m, ArH)

IR; 3328, 3035, 2955, 2629, 1732, 1452, 1391, 1214

(3) Troc-L-glutamic acid a-tert-butyl ester (24)

Under ice cooling, a commercially available (manufactured by WATANABECHEMICAL INDUSTRIES, LTD.) L-glutamic acid tert-butyl ester 23 (1.00 g,4.92 mmol) was dissolved in an aqueous solution (10 mL) of sodiumhydrogen carbonate (827 mg, 9.84 mmol), and a diethyl ether (3 mL)solution of Troc-Cl (0.80 mL, 5.97 mmol) was added dropwise thereto. Thereaction treatment was carried out in the same manner as in 22a toobtain a colorless oily matter 24 (1.61 g, 86.4%). This oily matter wasused for the next reaction without purification.

TLC; Rf=0.56 (A)

¹H-NMR (CDCl₃); 1.49 (9H, s), 1.92-2.08 (1H, m), 2.18-2.30 (1H, m),2.36-2.56 (2H, m), 4.27-4.36 (1H, m), 4.64-4.81 (2H, m), 4.73 (1H, d,J=7.81 Hz)

¹³C-NMR (CDCl₃); 27.51, 27.93, 29.79, 53.79, 74.61, 83.03, 95.29,154.21, 170.52, 178.12

IR; 3336, 2980, 1720, 1528, 1452, 1395, 1370, 1230

(4) Troc-L-glutamic acid (AHA-OBn) a-tert-butyl ester (25)

Under ice cooling, an acetonitrile (7 mL) solution of 20 p-Tos-OH salt(1.83 g, 4.65 mmol) was neutralized by adding DIPEA (1.84 mL, 10.6mmol), then an acetonitrile solution (3 mL) of 24 (1.60 g, 4.23 mmol)was added thereto, and EDC hydrochloride (972 mg, 5.04 mmol) and HOBt(571 mg, 4.23 mmol) were subsequently added. The ice cooling bath wasremoved, and the reaction liquid was stirred under room temperatureovernight, and then the solvent was distilled away under reducedpressure. This residue was dissolved in ethyl acetate, and the organiclayer was washed with a 10% aqueous solution of citric acid, a saturatedaqueous solution of sodium hydrogen carbonate and a saturated salinesolution, dried over anhydrous sodium sulfate, and concentrated underreduced pressure. This residue was purified by silica gel columnchromatography (chloroform) to obtain a colorless oily matter 25 (1.82g, 74%).

TLC; Rf=0.69 (B)

¹H-NMR (CDCl₃); ¹H-NMR (CDCl₃); 1.30-1.40 (2H, m), 1.43-1.56 (11H, m),1.62-1.70 (2H, m), 1.91-2.03 (1H, m), 2.17-2.30 (3H, m), 4.37 (2H, t,J=7.32 Hz), 3.17-3.31 (2H, m), 4.17-4.27 (1H, m), 4.63-4.81 (2H, m),5.11 (2H, s), 5.90 (1H, d, J=7.56 Hz), 7.31-7.39 (5H, m)

¹³C-NMR (CDCl₃); 24.36, 26.25, 27.91, 28.71, 29.06, 32.42, 34.00, 39.31,54.15, 66.13, 74.54, 82.71, 95.33, 128.16, 128.18, 128.51, 135.91,154.56, 170.61, 171.68, 173.39

IR; 3322, 3034, 2938, 2867, 1734, 1651, 1537, 1454, 1369, 1229

(5) Troc-L-glutamic acid (AHA-OBn) (22b)

Under ice cooling, trifluoroacetic acid (5.0 mL) was added to adichloromethane (5.0 mL) solution of 25 (913 mg, 1.57 mmol), and themixture was stirred for 30 minutes, and subsequently stirred under roomtemperature for 2.5 hours. After confirming completion of the reactionby TLC, the reaction liquid was concentrated under reduced pressure toobtain a pale yellow oily matter 22b TFA salt (825 mg, quant.). Thisoily matter was used for the next reaction without purification.

TLC; Rf=0.50 (C)

¹H-NMR (CDCl₃); 1.30-1.39 (2H, m, CH₂), 1.46-1.57 (2H, m, CH₂),1.59-1.70 (2H, m, CH₂ ), 2.06-2.14 (1H, m, CHCH₂ CH₂CO), 2.18-2.29 (1H,m, CHCH₂ CH₂CO), 2.34-2.46 (4H, m, CH₂), 3.20-3.30 (2H, m, CH₂),4.32-4.39 (1H, m, CHCH₂CH₂CO), 4.64-4.90 (2H, m, CH₂ CCl₃), 5.12 (2H, s,CH₂ Ph), 6.28 (1H, d, J=7.32, NH), 6.36-6.41 (1H, m, NH), 7.30-7.40 (5H,m, ArH)

¹³C-NMR (CDCl₃); 24.24, 26.15, 28.68, 28.99, 32.43, 33.98, 39.71, 54.43,66.35, 74.65, 95.32, 128.22, 128.30, 128.60, 135.88, 154.52, 172.92,173.73, 173.76

[a]_(D)+8.57° (CHCl₃, c 0.46, 16.4° C.)

IR; 3341, 2942, 2602, 1733, 1626, 1541, 1450, 1230, 736

D-Leucine Acid and D-Valine Acid Tert-Butyl Esters

(6) D-OAc-leucine {(2R)-2-acetoxy-4-methylpentanoic acid} (27)

Under ice cooling, acetyl chloride (13.4 mL, 154 mmol) was added toD-O-leucine 26 (3, 3.36 g, 25.4 mmol) using a dropping funnel. Afterconfirming disappearance of the raw material by TLC, the reaction liquidwas concentrated under reduced pressure. In order to completely removeacetyl chloride in this concentrated residue, operation ofco-evaporation with a small amount of toluene was repeated three timesto obtain a colorless oily matter 27 (6.52 g, quant.). This oily matterwas used for the reaction without purification.

TLC; Rf=0.61 (C)

¹H-NMR (CDCl₃); 0.94 (3H, d, J=6.34, CH(CH₃ )₂), 0.97 (3H, d, J=6.34,CH(CH₃ )₂) 1.64-1.72 (1H, m, CH(CH₃)₂), 1.75-1.86 (2H, m, CH₂ CH(CH₃)₂),2.15 (3H, s, COCH₃ ), 5.01-5.07 (1H, m, CHCH₂)

¹³C-NMR (CDCl₃); 20.53, 21.41, 22.91, 24.57, 39.50, 70.55, 170.79,176.70

(7) D-OAc-leucine tert-butyl ester {tert-butyl(2R)-2-acetoxy-4-methylpentanoate} (28)

Under room temperature, DMAP (0.92 g, 7.53 mmol) was slowly added to amixture of 27 (6.52 g, 37.4 mmol), tert-butanol (40 mL) and Boc₂O (8.26g, 37.8 mmol). After stirring the mixture for 5 hours at roomtemperature, the reaction liquid was concentrated under reducedpressure, and the concentrated residue was purified by silica gel columnchromatography (hexane/ethyl acetate) to obtain a colorless oily matter28 (6.56 g, 70.1%).

TLC; Rf=0.54 (A)

¹H-NMR (CDCl₃); 0.92 (3H, d, J=6.83, CH(CH₃ )₂), 0.96 (3H, d, J=6.58 Hz,CH(CH₃ )₂), 1.46 (9H, s, tert-Bu), 1.55-1.64 (1H, m, CH(CH₃)₂),1.69-1.82 (2H, m, CH₂ CH(CH₃)₂), 2.12 (3H, s, COCH₃ ), 4.85-4.92 (1H, m,CHCH₂)

¹³C-NMR (CDCl₃); 20.70, 21.56, 22.99, 24.62, 27.91, 39.71, 71.52, 81.89,169.94, 170.64

IR; 2963, 2875, 1746, 1465, 1372

(8) D-O-leucine tert-butyl ester {tert-butyl(2R)-2-hydroxy-4-methylpentanoate} (29)

An aqueous solution (60 mL) of potassium carbonate (19.68 g, 142.4 mmol)was added to a methanol (30 mL) solution of D-OAc-leucine tert-butylester (6.56 g, 28.5 mmol), and the mixture was vigorously stirred atroom temperature for one day. After completion of the reaction, methanolwas distilled away under reduced pressure, and the residue wastransferred to a separating funnel and extracted three times withdiethyl ether. The combined organic layer was washed with a saturatedsaline solution, then dried over anhydrous sodium sulfate, andconcentrated under reduced pressure, to obtain a colorless oily matterD-O-leucine tert-butyl ester (4.35 g, 81.1%). This oily matter was usedfor the next reaction without purification.

TLC; Rf=0.52 (A)

¹H-NMR (CDCl₃); 0.94 (3H, d, J=2.68 Hz, CH(CH₃ )₂), 0.96 (3H, d, J=2.44Hz, CH(CH₃ )₂), 1.49-1.53 (11H, m, tert-Bu and CHCH₂ CH), 1.81-1.95 (1H,m, CH(CH₃)₂), 2.74 (1H, d, J=5.85 Hz, OH), 4.00-4.09 (1H, m,CHCH₂(CH₃)₂)

¹³C-NMR (CDCl₃); 21.59, 23.32, 24.49, 27.99, 43.59, 69.20, 82.23, 175.23

[a]_(D)−6.90° (CHCl₃, c 1.06, 14.7° C.)

IR; 3493, 2959, 2873, 1729, 1465, 1273

(9) L-OAc-valine {(2R)-2-acetoxy-4-methylpentanoic acid} (30)

L-OAc-valine was synthesized in the same manner as that forD-OAc-leucine. More specifically, acetyl chloride (15.0 mL, 211 mmol)was added from a dropping funnel to L-O-valine 5 (5.00 g, 42.3 mmol)under ice cooling. After confirming disappearance of the raw material byTLC, the reaction liquid was concentrated under reduced pressure. Asmall amount of toluene was added to this residue, the mixture wasconcentrated under reduced pressure, and the remaining acetyl chloridewas removed by co-evaporation with toluene. This operation was repeatedthree times to obtain a colorless oily matter 30 (6.73 g, quant) as aconcentrated residue. This oily matter was used for the next reactionwithout purification.

TLC; Rf=0.63 (C)

¹H-NMR (CDCl₃); 1.00-1.06 (6H, m, CH(CH₃ )₂), 2.16 (3H, s, CH₂ CO),2.20-2.33 (1H, m, CH(CH₃)₂), 4.89 (1H, d, J=4.39 Hz, CHCH(CH₃)₂), 10.64(1H, br, COOH)

¹³C-NMR (CDCl₃); 17.03, 18.70, 20.48, 29.86, 170.92, 175.61

(10) L-OAc-valine tert-butyl ester {tert-butyl(2S)-2-acetoxy-3-methylbutanoate} (31)

L-OAc-valine tert-butyl ester was synthesized in the same manner as thatfor D-OAc-leucine tert-butyl ester. More specifically, a mixture ofL-OAc-valine (6.73 g, 42.0 mmol), tert-butanol (40 mL) and Boc₂O (9.17g, 42.0 mmol) was stirred under room temperature, and DMAP (1.03 g, 8.43mmol) was slowly added thereto. After stirring the mixture at roomtemperature for 2 hours, the reaction liquid was concentrated underreduced pressure, and the concentrated residue was purified by silicagel column chromatography (hexane/ethyl acetate) to obtain a colorlessoily matter L-OAc-valine tert-butyl ester (6.52 g, 71.8%).

TLC; Rf=0.57 (A)

¹H-NMR (CDCl₃); 0.95-1.01 (6H, m, CH(CH₃ )₂), 1.47 (9H, s, tert-Bu),2.17 (3H, s, CH₂ CO), 2.16-2.24 (1H, m, CH(CH₃)₂), 4.72 (1H, d, J=4.39Hz, CHCH(CH₃)₂)

¹³C-NMR (CDCl₃); 17.12, 18.71, 20.64, 27.96, 29.93, 60.37, 81.83,168.78, 170.77

IR; 2975, 2938, 2880, 1744, 1238

(11) L-O-valine tert-butyl ester {tert-butyl(2S)-2-hydroxy-3-methylbutanoate} (32)

L-O-valine tert-butyl ester was synthesized in the same manner as thatfor L-O-leucine tert-butyl ester. More specifically, an aqueous solution(60 mL) of potassium carbonate (20.83 g, 151.0 mmol) was added to amethanol (30 mL) solution of L-OAc-valine tert-butyl ester (6.52 g, 30.1mmol), and the mixture was stirred at room temperature for one day.After completion of the reaction, methanol was distilled away underreduced pressure, and the aqueous layer was extracted three times withdiethyl ether. The combined organic layer was washed with a saturatedsaline solution, then dried over anhydrous sodium sulfate, andconcentrated under reduced pressure, to obtain a colorless oily matterL-O-valine tert-butyl ester (3.87 g, 73.7%). This oily matter was usedfor the next reaction without further purification.

TLC; Rf=0.54 (A)

¹H-NMR (CDCl₃); 0.86 (3H, d, J=6.83 Hz, CH(CH₃ )₂), 1.02 (3H, d, J=7.07Hz, CH(CH₃ )₂), 1.50 (9H, s, tert-Bu), 3.92 (1H, d, J=3.41, CHCH(CH₃)₂)

¹³C-NMR (CDCl₃); 15.72, 18.80, 25.58, 28.02, 32.08, 74.88, 82.31, 174.22

[a]_(D)+3.60° (MeOH, c 1.04, 15.1° C.)

IR; 3518, 2970, 2879, 1726, 1465, 1259

Example 7

Synthesis of Didepsipeptides

(12) Troc-L-glutamic acid (OBn/AHA-OBn)-D-O-leucine tert-butyl ester(33)

Under ice cooling, DMAP (0.34 mmol) was slowly added to adichloromethane (10 mL) solution of N-protected L-glutamic acidderivative 22 (1.12 mmol) and D-leucine tert-butyl ester 29 (1.35 mmol),and then DCC (1.68 mmol) was slowly added thereto. This reaction liquidwas stirred under ice cooling overnight, then the by-produced DCUrea wasremoved by suction filtration, and the filtrate was concentrated underreduced pressure. This concentrated residue was dissolved in ethylacetate, and the organic layer was washed with a saturated aqueoussolution of sodium hydrogen carbonate, followed by a saturated salinesolution, dried over anhydrous sodium sulfate, and concentrated underreduced pressure. The concentrated residue was purified by silica gelcolumn chromatography (hexane/ethyl acetate) to obtain a targetdidepsipeptide 33.

33a (R═OBn);

Yield; 35%

TLC; Rf=0.60 (F)

¹H-NMR (CDCl₃); 0.91 (3H, d, J=6.59 Hz), 0.94 (3H, d, J=6.34 Hz), 1.45(9H, s), 1.57-1.67 (2H, m), 1.70-1.81 (1H, m), 2.04-2.18 (1H, m,),2.28-2.43 (1H, m), 2.47-2.61 (2H, m), 4.51-4.60 (1H, m), 4.67-4.76 (2H,m), 4.89-4.94 (1H, m), 5.13 (2H, s), 5.69 (1H, d, J=8.29 Hz), 7.30-7.40(5H, m)

¹³C-NMR (CDCl₃); 21.40, 23.01, 24.62, 26.97, 27.32, 27.88, 29.99, 39.46,53.53, 66.59, 72.69, 74.45, 74.62, 82.43, 95.24, 128.22, 128.31, 128.57,135.62, 154.04, 168.94, 172.38

IR; 3340, 2959, 1739, 1525, 1453, 1389, 1370, 1259, 1206

33b (R═NH(CH₂)₅COOBn);

Yield; 93%

TLC; Rf=0.42 (F)

¹H-NMR (CDCl₂); 0.89-0.95 (6H, m, CH(CH₃ )₂), 1.30-1.40 (2H, m, CH₂),1.46 (9H, s, tert-Bu), 1.47-1.55 (2H, m, CH₂), 1.59-1.80 (6H, m),2.06-2.16 (1H, m, CHCH₂ CH₂CO), 2.28-2.48 (5H, m), 3.16-3.28 (2H, m,CH₂), 4.39-4.47 (1H, m, CHCH₂CH(CH₃)₂), 4.66-4.77 (2H, m, CH₂ CCl₃),4.90-4.95 (1H, m, NH), 5.11 (2H, s, CH₂Ph), 6.07-6.11 (2H, m, NH),7.33-7.37 (5H, m, ArH)

¹³C-NMR (CDCl₃); 14.18, 21.05, 21.40, 23.03, 24.41, 24.55, 26.32, 27.90,28.01, 29.07, 32.23, 34.04, 39.38, 39.57, 54.11, 60.39, 66.17, 72.49,74.61, 82.57, 95.29, 128.20, 128.54, 129.01, 135.96, 154.37, 169.54,170.86, 171.97, 173.38

IR; 3322, 2956, 2871, 1741, 1653, 1537, 1239, 735

(13) Troc-L-glutamic acid (OBn/AHA-OBn)-D-O-leucine (34)

Under ice cooling, TFA (3.0 mL) was slowly added to a dichloromethane(3.0 mL) solution of 33 (0.85 mmol), and the mixture was stirred for 30minutes under ice cooling, and subsequently stirred under roomtemperature for 1.5 hours. After confirming completion of the reactionby TLC, the reaction liquid was concentrated under reduced pressure toobtain a pale yellow oily matter 34 (quant.). This oily matter was usedfor the next reaction without purification.

34a (R═OBn)

Yield; quant

TLC; Rf=0.56 (C)

¹H-NMR (CDCl₃); 0.92 (3H, d, J=6.10 Hz), 0.95 (3H, d, J=6.34 Hz),1.65-1.78 (2H, m), 1.80-1.91 (1H, m), 2.02-2.16 (1H, m), 2.26-2.38 (1H,m), 2.41-2.60 (2H, m), 4.49-4.58 (1H, m), 4.62-4.78 (2H, m), 5.06-5.12(1H, m), 5.13 (2H, s), 5.74 (1H, d, J=8.05 Hz), 7.29-7.40 (5H, m)

¹³C-NMR (CDCl₃); 21.29, 22.96, 24.62, 27.11, 29.97, 39.34, 53.54, 66.70,71.61, 74.70, 95.19, 128.26, 128.36, 128.60, 135.55, 154.19, 170.84,172.50

IR; 3419, 2959, 1736, 1643, 1521, 1452, 1391, 1210, 1099

34b (R═NH(CH₂)₅COOBn)

Yield; quant

TLC; Rf=0.52 (C)

¹H-NMR (CDCl₃); 0.88-0.99 (6H, m, CH(CH₃ )₂), 1.29-1.40 (2H, m, CH₂),1.44-1.56 (2H, m, CH₂), 1.60-1.67 (2H, m, CH₂), 1.68-1.79 (2H, m, CH₂),1.82-1.88 (1H, m, CH(CH₃)₂), 2.10-2.19 (1H, m, CHCH₂ CH₂CO), 2.19-2.30(1H, m, CHCH₂ CH₂CO), 2.35-2.42 (4H, m, CH₂), 3.19-3.28 (2H, m, CH₂),4.39-4.47 (1H, m, CHCH₂CH(CH₃)₂), 4.67-4.77 (2H, m, CH₂ CCl₃), 5.09-5.11(1H, m, NH), 5.12 (2H, s, CH₂Ph), 6.29 (1H, d, J=7.56 Hz, NH), 6.45-6.52(1H, m, NH), 7.33-7.39 (5H, m, ArH), 8.77 (1H, br, COOH)

¹³C-NMR (CDCl₃); 21.30, 23.03, 24.34, 24.58, 26.17, 28.01, 28.67, 32.07,34.05, 39.36, 39.53, 54.08, 66.47, 71.82, 74.64, 95.22, 128.21, 128.31,128.58, 135.66, 154.56, 170.81, 172.67, 173.18, 174.17

IR; 3351, 3062, 2957, 2871, 1734, 1630, 1539, 1453, 1386, 1211, 1099

(14) Carbobenzyloxy-D-alanine (Z-D-Ala)

Under ice cooling, commercially available D-alanine (6.00 g, 73.5 mmol)was dissolved in an aqueous solution (225 mL) of sodium carbonate (21.45g, 202 mmol), and a dioxane (60 mL) solution of benzyl chloroformate(Z-Cl) (10.5 mL, 73.5 mmol) was slowly added dropwise thereto. Aftercompletion of the dropwise addition, the reaction liquid was returned toroom temperature, and continuously stirred overnight. The reactionliquid was transferred to a separating funnel, and excess Z-Cl wasremoved by diethyl ether extraction. Citric acid was added to theaqueous layer to adjust the pH to 3, and then the mixture was extractedthree times with ethyl acetate. The combined organic layer was washedonce with a saturated saline solution, and the organic layer was driedover anhydrous sodium sulfate, and concentrated under reduced pressure,and then the concentrated residue was recrystallized by ethylacetate-hexane to obtain a colorless crystal Z-D-alanine (13.77 g,91.6%).

TLC; Rf=0.51 (C)

mp; 82.9° C.

¹H-NMR (CDCl₃); 1.47 (3H, d, J=7.32 Hz, CH₃ ), 4.38-4.48 (1H, m, CHCH₃),5.13 (2H, s, CH₂ Ph), 5.29 (1H, br, NH), 7.30-7.38 (5H, m, ArH)

¹³C-NMR (CDCl₃); 18.36, 49.52, 67.22, 128.10, 128.25, 128.53, 136.05,155.92, 177.45

IR; 3349, 3046, 1719, 1539, 1252

(15) Z-D-Alanine-L-O-valine tert-butyl ester (35)

Under ice cooling, DMAP (0.35 g, 2.9 mmol) was slowly added to adichloromethane (30 mL) solution of L-O-valine tert-butyl ester 32 (2.50g, 14.3 mmol) and Z-D-alanine (3.52 g, 15.8 mmol), and then DCC (3.70 g,17.9 mmol) was slowly added thereto. This reaction liquid was stirredunder ice cooling overnight, then the by-produced DCUrea was removed bysuction filtration, and the filtrate was concentrated under reducedpressure. This concentrated residue was dissolved in ethyl acetate, andthe organic layer was washed with a saturated aqueous solution of sodiumhydrogen carbonate, followed by a saturated saline solution, dried overanhydrous sodium sulfate, and concentrated under reduced pressure. Thisresidue was purified by silica gel column chromatography (hexane/ethylacetate) to obtain a colorless oily matter 35 (4.26 g, 78.3%).

TLC; Rf=0.36 (A)

¹H-NMR (CDCl₃); 0.92-1.02 (6H, m, CH(CH₃ )₂), 1.46 (9H, s, tert-Bu),1.47 (3H, d, J=7.07 Hz, CHCH₃ ), 2.18-2.29 (1H, m, CH(CH₃)₂), 4.46-4.57(1H, m, CHCH₃), 4.75 (1H, d, J=4.15 Hz, CHCH(CH₃)₂), 5.11 (2H, s, CH₂Ph), 5.35 (1H, d, J=7.07 Hz, NH), 7.29-7.40 (5H, m, ArH)

¹³C-NMR (CDCl₃); 16.93, 18.73, 27.93, 30.00, 49.74, 66.84, 82.22,128.11, 128.13, 128.50, 136.23, 155.46, 168.05, 172.34

IR; 3342, 2974, 2937, 2879, 1734, 1528, 1254

[a]_(D)−18.04° (CHCl₃, c 0.875, 15.7° C.)

(16) D-Alanine-L-O-valine tert-butyl ester (36) TFA Salt

A flask for hydrogenation was charged with a mixture of 35 (1.96 g, 5.17mmol), methanol (30 mL), TFA (0.46 mL, 6.2 mmol) and 10% palladiumcarbon (100 mg), and the mixture was stirred under a hydrogen stream (3atmospheres) at room temperature for 3 hours. After confirming theprogress of the reaction by TLC, the catalyst was removed by filtration,the filtrate was concentrated under reduced pressure, and theconcentrated residue was purified by silica gel column chromatography(chloroform/methanol) to obtain a light brown oily matter 36 (1.65 g,93.3%).

TLC; Rf=0.34 (B)

¹H-NMR (CDCl₃); 0.96-1.04 (6H, m, CH(CH₃ )₂), 1.36 (3H, d, J=8.54 Hz,CHCH₃ ), 1.47 (9H, s, tert-Bu), 2.20-2.29 (1H, m, CH(CH₃)₂), 3.66 (1H,q, J=7.07 Hz, CHCH₃), 4.76 (1H, d, J=4.39 Hz, CHCH(CH₃)₂)

¹³C-NMR (CDCl₃); 16.99, 18.80, 20.29, 27.97, 28.01, 30.02, 32.07, 49.97,82.01, 168.52, 176.24

IR; 3445, 2977, 1747, 1677, 1537, 1464, 1432, 1395, 1371, 1330, 1204

Example 8

Synthesis of Tetradepsipeptides

(17) Troc-L-glutamic acid (OBn/AHA-OBn)-D-O-leucine-D-alanine-L-O-valinetert-butyl ester (37)

Under ice cooling, an acetonitrile (5 mL) solution of N-terminalprotected didepsipeptide 36 TFA salt (289 mg, 0.84 mmol) was neutralizedby adding DIPEA (0.15 mL, 0.84 mmol), then an acetonitrile (5 mL)solution of C-terminal protected didepsipeptide 34 (0.84 mmol) wasadded, and HBTU (352 mg, 0.93 mmol) and HOBt (114 mg, 0.84 mmol) weresubsequently added. The reaction liquid was returned to room temperatureand stirred overnight, and then the reaction liquid was concentratedunder reduced pressure. This concentrated residue was dissolved in ethylacetate, and the ethyl acetate layer was sequentially washed with a 10%aqueous solution of citric acid, a saturated aqueous solution of sodiumhydrogen carbonate and a saturated saline solution, and dried overanhydrous sodium sulfate, and then concentrated under reduced pressure.This residue was purified by silica gel column chromatography(hexane/ethyl acetate) to obtain a pale yellow oily matter 37.

37a (R═OBn);

Yield; 75%

TLC; Rf=0.64 (F)

¹H-NMR (CDCl₃); 0.85-1.00 (12H, m), 1.45 (9H, s), 1.48 (3H, d, J=7.32Hz), 1.60-1.83 (3H, m), 1.99-2.15 (1H, m), 2.15-2.36 (2H, m), 2.41-2.62(2H, m), 4.43-4.52 (1H, m), 4.60-4.81 (4H, m), 5.14 (2H, s), 5.27 (1H,dd, J=5.61 Hz, J=7.56 Hz), 5.98 (1H, d, J=7.56 Hz), 6.83 (1H, d, J=7.56Hz), 7.30-7.40 (5H, m)

¹³C-NMR (CDCl₃); 16.88, 17.72, 18.69, 21.42, 23.09, 24.47, 26.52, 27.94,30.02, 40.50, 47.95, 53.69, 66.73, 73.49, 74.74, 82.33, 95.18, 128.28,128.38, 128.59, 135.49, 154.50, 168.28, 169.22, 170.47, 171.91, 172.37

IR; 3421, 2964, 1739, 1674, 1538, 1455, 1391, 1370, 1256

37b (R═NH(CH₂)₅CO₂Bn);

Yield; 55%

TLC; Rf=0.31 (F)

¹H-NMR (CDCl₃); 0.85-1.00 (12H, m), 1.30-1.40 (2H, m), 1.45 (9H, s),1.49 (3H, d, J=7.32 Hz), 3.19-3.29 (1H, m), 4.39-4.46 (1H, m), 4.62-4.80(4H, m), 5.11 (2H, s), 5.23 (1H, dd, J=4.88 Hz, J=9.02 Hz), 6.03-6.11(1H, m), 6.59 (1H, d, J=7.07 Hz), 7.00 (1H, d, J=7.56 Hz), 7.30-7.40(5H, m)

¹³C-NMR (CDCl₃); 16.89, 17.59, 18.69, 21.39, 23.12, 24.33, 24.41, 26.25,26.92, 27.92, 28.96, 30.00, 31.97, 33.99, 39.45, 40.46, 48.12, 54.22,66.17, 73.24, 74.66, 77.68, 82.28, 95.27, 128.17, 128.22, 128.54,128.55, 135.90, 154.72, 168.24, 169.68, 170.68, 171.96, 172.26, 173.44

IR; 3335, 2962, 1757, 1680, 1538, 1455, 1108, 736

(18) L-Glutamic acid (OBn/AHA-OBn)-D-O-leucine-D-alanine-L-O-valinetert-butyl ester (38)

Zinc (310.6 mg, 4.74 mmol) was added to a 90% acetic acid solution (3mL) of C- and N-terminal protected tetradepsipeptide 37 (0.24 mmol), andthe mixture was stirred at room temperature for one day. Aftercompletion of the reaction, a large amount of water was added to thereaction liquid, and the mixture was extracted three times with ethylacetate. The combined organic layer was washed with water, a saturatedaqueous solution of sodium hydrogen carbonate, and a saturated salinesolution. The organic layer was dried over anhydrous sodium sulfate, andconcentrated under reduced pressure to obtain 38.

38a (R═OBn); Pale Yellow Oily Matter;

Yield; quant

TLC; Rf=0.53 (B)

¹H-NMR (CDCl₃); 0.78-1.03 (12H, m), 1.40-1.50 (12H, m), 1.60-1.82 (3H,m), 2.05-2.32 (3H, m), 2.31-2.59 (2H, m), 4.33-4.45 (1H, m), 4.65-4.81(2H, m), 5.13 (2H, s), 5.18-5.25 (1H, m), 6.57 (1H, d, J=7.56 Hz), 6.88(1H, d, J=7.32 Hz), 7.29-7.38 (5H, m)

¹³C-NMR (CDCl₃); 16.94, 18.20, 18.74, 21.53, 23.13, 24.53, 27.96, 27.97,30.05, 40.64, 47.72, 55.22, 66.39, 72.93, 77.24, 77.83, 82.38, 126.99,128.20, 128.57, 135.83, 168.13, 168.97, 169.65, 172.22, 172.89

IR; 3346, 2965, 2938, 2875, 1740, 1679, 1538, 1455, 1391, 1370

38b (R═NH(CH₂)₅COOBn); Pale Yellow Amorphous Solid;

Yield; 96%

TLC; Rf=0.42 (C)

¹H-NMR (CDCl₃); 0.82-1.02 (12H, m), 1.30-1.39 (2H, m), 1.42-1.55 (15H,m), 1.58-1.84 (5H, m), 1.87-1.99 (1H, m), 2.14-2.29 (2H, m), 2.29-2.42(4H, m), 3.13-3.29 (2H, m), 3.63-3.76 (1H, m), 4.70 (1H, d, J=7.56 Hz),4.73 (1H, d, J=4.39 Hz), 5.11 (2H, s), 5.23 (1H, dd, J=3.90 Hz, J=9.51Hz), 6.12-6.24 (1H, m), 7.04 (1H, d, J=7.56 Hz), 7.29-7.38 (5H, m)

¹³C-NMR (CDCl₃); 14.16, 16.96, 18.00, 18.69, 21.47, 23.10, 24.42, 24.45,26.32, 27.90, 29.14, 30.01, 32.42, 34.02, 39.25, 40.64, 47.76, 60.39,66.14, 73.02, 82.39, 128.17, 128.20, 128.53, 135.91, 168.19, 169.73,172.15, 172.29, 173.41

IR; 3317, 3068, 2962, 2873, 1739, 1663, 1637, 1545, 1456, 1370, 1163

Example 9

Synthesis of Dodecadepsipeptides (Precursors)

(19)Boc-L-valine-D-O-leucine-D-alanine-L-O-valine-L-valine-D-O-leucine-D-alanine-L-O-valine-L-glutamicacid (OBn/AHA-OBn)-D-O-leucine-D-alanine-L-O-valine tert-butyl ester(39)

Under ice cooling, an acetonitrile (5 mL) solution of C-terminalprotected tetradepsipeptide 38 (0.155 mmol) and N-terminal protectedoctadepsipeptide 15 (137 mg, 0.155 mmol) was charged, and DIPEA (27.0μL, 0.155 mmol), HOAt (21.1 mg, 0.155 mmol) and HATU (88.2 mg, 0.232mmol) were added thereto. The reaction liquid was returned to roomtemperature and stirred overnight, concentrated under reduced pressure,then the residue was dissolved in ethyl acetate, and the ethyl acetatelayer was sequentially washed with a 10% aqueous solution of citricacid, a saturated aqueous solution of sodium hydrogen carbonate and asaturated saline solution. The organic layer was dried over anhydroussodium sulfate, and then concentrated under reduced pressure, and theresidue was purified by silica gel column chromatography (hexane/ethylacetate=3:2) to obtain a colorless oily matter 39.

39a (R═OBn);

Yield; 40%

TLC; Rf=0.64 (F)

¹H-NMR (CDCl₃); 0.74-1.09 (48H, m), 1.32-1.53 (27H, m), 1.60-1.86 (9H,m), 1.92-2.04 (1H, m), 2.13-2.26 (2H, m), 2.27-2.44 (4H, m), 2.45-2.52(2H, m), 3.84 (1H, dd, J=5.85 Hz, J=7.56 Hz), 4.01-4.16 (2H, m), 4.27(1H, t, J=7.56 Hz), 4.48-4.56 (1H, m), 4.60 (1H, t, J=7.56 Hz), 4.65(1H, d, J=4.15 Hz), 4.97 (1H, d, J=2.93 Hz), 5.07 (1H, d, J=2.68 Hz),5.27 (1H, dd, J=3.90 Hz, J=8.87 Hz), 7.28-7.37 (5H, m), 7.74 (1H, d,J=7.32 Hz), 7.87 (1H, d, J=7.32 Hz), 7.96 (1H, d, J=5.85 Hz), 8.10 (1H,d, J=7.81 Hz), 8.25 (1H, d, J=6.10 Hz)

¹³C-NMR (CDCl₃); ¹³C-NMR (CDCl₃); 16.18, 16.34, 16.56, 17.02, 17.57,18.67, 18.95, 19.00, 19.06, 19.22, 19.30, 20.78, 20.89, 21.17, 23.12,23.13, 23.36, 24.29, 24.39, 24.42, 26.20, 27.93, 28.26, 29.66, 29.80,29.98, 30.08, 30.19, 30.41, 40.53, 40.65, 40.76, 48.25, 49.43, 49.63,52.06, 58.97, 60.40, 66.31, 72.69, 72.75, 73.04, 77.56, 78.65, 79.00,81.00, 81.79, 128.09, 128.24, 128.47, 135.93, 156.56, 168.44, 169.99,170.15, 170.44, 170.90, 170.93, 171.92, 172.19, 172.26, 172.44, 172.50

IR; 3316, 2965, 2876, 1748, 1658, 1538, 1460, 1369, 1158

38b (R═NH(CH₂)₅COOBn);

Yield; 73.5%

TLC; Rf=0.58 (B)

¹H-NMR (CDCl₃); 0.82-1.09 (48H, m), 1.39-1.53 (33H, m), 1.53-1.89 (11H,m), 1.93-2.05 (1H, m), 2.10-2.44 (9H, m), 3.09-3.28 (2H, m), 3.84 (1H,dd, J=5.85 Hz, J=7.56 Hz), 4.03-4.19 (2H, m), 4.30 (1H, t, J=7.56 Hz),4.37-4.48 (1H, m), 4.59-4.67 (2H, m), 4.97 (1H, d, J=3.17 Hz), 5.02 (1H,d, J=2.93 Hz), 5.07-5.15 (4H, m), 5.20-5.30 (1H, m), 6.53 (1H, s),7.28-7.40 (5H, m), 7.66 (1H, d, J=7.32 Hz), 7.87 (1H, d, J=7.56 Hz),7.94-8.01 (2H, m), 8.29 (1H, d, J=6.10 Hz)

¹³C-NMR (CDCl₃); 16.22, 16.33, 16.52, 17.07, 17.62, 18.63, 18.90, 18.96,19.05, 19.20, 20.77, 20.85, 21.14, 23.06, 23.11, 23.33, 24.29, 24.37,24.56, 26.46, 27.46, 27.90, 27.93, 28.24, 29.28, 29.69, 29.78, 29.97,30.03, 30.16, 32.48, 34.13, 39.36, 40.50, 40.66, 40.75, 48.04, 49.28,49.63, 52.21, 58.84, 60.39, 66.05, 72.70, 72.77, 73.06, 77.20, 77.54,77.77, 78.82, 78.98, 80.96, 81.93, 128.13, 128.18, 128.50, 136.03,156.51, 168.31, 169.85, 170.35, 170.59, 170.86, 170.91, 170.98, 171.87,172.14, 172.23, 172.47, 173.45

IR; 3319, 2965, 2875, 1748, 1656, 1539, 1460, 1370, 1158, 747

(20)L-Valine-D-O-leucine-D-alanine-L-O-valine-L-valine-D-O-leucine-D-alanine-L-O-valine-L-glutamicacid (OBn/AHA-OBn)-D-O-leucine-D-alanine-L-O-valine (40)

Under ice cooling, TFA (2 mL) was slowly added to a dichloromethane (2mL) solution of 39 (69 μmol), and the mixture was stirred under icecooling for 1 hour. After confirming the progress of the reaction byTLC, toluene was added to the reaction liquid, and the mixture wasconcentrated under reduced pressure. In order to completely remove TFA,toluene was added to the concentrated residue and TFA was azeotropicallydistilled away. This operation was repeated three times to obtain a paleyellow solid 40. This solid was used for the next cyclization reactionwithout purification.

40a (R═OBn);

Yield; 94%

TLC; Rf=0.43 (B)

¹H-NMR (CDCl₃); 0.83-1.09 (48H, m), 1.39 (3H, d, J=7.32 Hz), 1.47 (3H,d, J=7.07 Hz), 1.51 (3H, d, J=7.56 Hz), 1.58-1.84 (9H, m), 2.10-2.41(9H, m), 4.10-4.25 (2H, m), 4.26-4.38 (2H, m), 4.57-4.66 (1H, m),4.73-4.79 (1H, m), 4.86 (1H, d, J=3.42 Hz), 4.93 (1H, d, J=2.93 Hz),5.02 (1H, s), 5.05-5.17 (3H, m), 5.27 (1H, dd, J=4.39 Hz, J=7.56 Hz),7.31-7.41 (5H, m), 7.64 (2H, d, J=8.54 Hz), 7.74 (1H, d, J=3.17 Hz),7.77 (1H, d, J=7.81 Hz), 8.32 (1H, d, J=5.61 Hz), 8.64 (1H, d, J=5.85Hz)

¹³C-NMR (CDCl₃); 14.98, 15.63, 16.31, 16.70, 17.05, 17.23, 17.44, 17.71,18.41, 18.54, 18.66, 18.93, 19.12, 19.58, 20.95, 21.11, 21.62, 22.99,23.10, 23.16, 23.22, 24.33, 24.48, 24.52, 27.84, 29.66, 30.35, 30.54,40.03, 40.14, 40.38, 50.09, 53.42, 58.74, 66.56, 73.94, 75.13, 78.02,79.18, 79.24, 82.03, 128.23, 128.58, 128.95, 135.53, 169.15, 170.45,171.34, 171.54, 171.82, 171.94, 172.12, 172.36, 173.42

IR; 3296, 3068, 2965, 2870, 1749, 1661, 1543, 1466, 1372, 1202

40b (R═NH(CH₂)₅COOBn);

Yield; quant

TLC; Rf=0.36 (B)

¹H-NMR (CDCl₃); 0.80-1.09 (48H, m), 1.24-1.33 (11H, m), 1.36-1.42 (4H,m), 1.43-1.52 (3H, m), 1.55 (2H, d, J=7.32 Hz), 1.58-1.88 (9H, m),2.12-2.40 (9H, m), 3.00-3.14 (2H, m), 3.16-3.30 (1H, m), 3.97 (1H, d,J=4.63 Hz), 4.05 (1H, dd, J=7.07 Hz, J=10.49 Hz), 4.60 (1H, t, J=7.56Hz), 4.65-4.79 (4H, m), 4.83 (1H, d, J=3.41 Hz), 4.88 (1H, d, J=2.68Hz), 5.02-5.09 (2H, m), 5.11 (2H, s), 5.33 (1H, dd, J=3.41 Hz, J=9.02Hz), 6.25 (1H, s), 7.29-7.39 (5H, m), 7.54-7.66 (2H, m), 7.82 (1H, d,J=7.56 Hz), 8.58 (1H, d, J=7.32 Hz), 9.27 (1H, s)

¹³C-NMR (CDCl₃); 15.37, 15.74, 16.60, 16.74, 16.86, 17.02, 17.39, 17.84,18.39, 18.46, 18.86, 19.18, 19.38, 20.99, 21.17, 21.28, 22.94, 23.00,23.24, 24.38, 24.44, 24.49, 26.29, 28.47, 29.04, 29.60, 29.65, 29.83,30.53, 32.26, 34.00, 39.60, 40.13, 40.40, 47.67, 49.33, 53.17, 58.71,66.22, 73.03, 75.19, 77.21, 78.90, 128.18, 128.21, 128.24, 128.55,129.01, 135.89, 169.18, 170.62, 170.78, 171.03, 171.59, 171.63, 172.24,172.69, 173.26, 173.46

IR; 3300, 3081, 2965, 2876, 1748, 1657, 1545, 1462, 1374, 1203

Example 10

E-Cereulide and EAHA-Cereulide (Cyclization Reaction)

(22) E-Cereulide-OBn (41a) and EAHA-cereulide-OBn (41b)

A cyclization reaction of a precursor 40 was carried out in the samemanner as in cereulide synthesis. More specifically, in a reactionvessel replaced with argon, diphenylphosphoryl azide (DPPA) (9.8 μL,45.5 μmol) was dissolved in anhydrous N,N-dimethylformamide (12 mL), andthen N,N-diisopropylethylamine (15.7 μL, 90 μmol) was slowly addedthereto to prepare a liquid A.

Separately, the precursor 40 (31 μmol) was dissolved in anhydrousN,N-dimethylformamide (8 mL) to prepare a liquid B.

Under room temperature, the liquid B was slowly added dropwise to theliquid A using a microsyringe over 1 hour or more, and continuouslystirred under room temperature for 10 days. The reaction liquid wasconcentrated under reduced pressure to distill dichloromethane away, andthe residue was dissolved in ethyl acetate, and sequentially washed witha saturated aqueous solution of sodium hydrogen carbonate, 1 Nhydrochloric acid and a saturated saline solution. The organic layer wasdried over anhydrous sodium sulfate, and concentrated under reducedpressure, and the residue was purified by silica gel columnchromatography (hexane/ethyl acetate) to obtain a pale yellow solid 41.

41a (R═OBn);

TLC; Rf=0.69 (F)

¹H-NMR (CDCl₃); 0.78-1.03 (48H, m), 1.40-1.48 (9H, m), 1.58-1.84 (11H,m), 2.10-2.60 (9H, m), 4.00-4.10 (2H, m), 4.25-4.60 (3H, m), 4.91-5.01(2H, m), 5.02 (1H, d, J=3.17 Hz), 5.10-5.14 (3H, m), 5.23 (1H, dd,J=3.90 Hz, J=5.37 Hz), 5.26-5.33 (2H, m), 7.31-7.37 (5H, m), 7.62 (1H,d, J=6.59 Hz), 7.74-7.80 (3H, m), 7.84 (1H, d, J=5.85 Hz), 7.96 (1H, d,J=7.56 Hz)

¹³C-NMR (CDCl₃); 15.72, 15.80, 16.77, 16.89, 18.46, 18.52, 18.65, 19.26,19.32, 21.11, 21.20, 21.73, 23.01, 23.32, 24.32, 24.36, 24.51, 28.52,28.72, 30.28, 30.39, 30.47, 30.57, 40.39, 48.57, 48.79, 49.03, 51.78,59.41, 59.55, 66.42, 72.58, 72.78, 73.30, 78.47, 78.64, 78.77, 128.20,128.23, 128.53, 169.92, 170.42, 170.63, 171.02, 171.06, 171.33, 171.43,171.53, 171.74, 171.84, 172.04, 172.18

IR; 3433, 3310, 2964, 2872, 1742, 1654, 1538, 1463, 1389, 1371, 1198,1160

[a]_(D)+7.17° (CHCl₃, c 1.20, 25° C.)

MALDI/TOFMS; 1295 [M+Na]⁻, 1311 [M+K]⁺

41b (R═NH(CH₂)₅COOBn)

TLC; Rf=0.23 (F)

mp; 64.7° C.

¹H-NMR (CDCl₃); 0.82-1.01 (42H, m), 1.04 (3H, d, J=6.34 Hz), 1.06 (3H,d, J=6.10 Hz), 1.29-1.39 (2H, m), 1.40-1.54 (11H, m), 1.60-1.72 (6H, m),1.72-1.82 (4H, m), 1.90-2.00 (1H, m), 2.03-2.14 (2H, m), 2.15-2.42 (9H,m), 3.09-3.19 (1H, m), 3.21-3.33 (1H, m), 4.03-4.12 (2H, m), 4.26-4.40(2H, m), 4.41-4.53 (2H, m), 4.97 (1H, d, J=3.17 Hz), 5.02-5.05 (2H, m),5.11 (2H, s), 5.15-5.22 (1H, m), 5.25-5.33 (2H, m), 6.42-6.47 (1H, m),7.29-7.40 (5H, m), 7.63-7.77 (3H, m), 7.84 (1H, d, J=7.32 Hz), 7.91 (1H,d, J=7.07 Hz)

¹³C-NMR (CDCl₃); 15.91, 16.70, 16.99, 18.47, 18.56, 19.07, 19.21, 19.30,19.35, 21.04, 21.36, 21.50, 23.07, 23.27, 23.34, 24.34, 24.52, 26.41,28.61, 28.74, 29.29, 30.29, 30.35, 30.49, 32.51, 34.09, 39.34, 40.27,40.53, 48.31, 48.62, 48.90, 52.38, 59.10, 59.59, 66.11, 72.82, 73.17,78.20, 78.61, 78.89, 128.16, 128.52, 135.97, 170.31, 170.43, 170.62,170.89, 171.06, 171.25, 171.53, 171.68, 171.76, 173.36

IR; 3298, 2963, 1744, 1657, 1538, 1461

MALDI/TOFMS; 1409 [M+Na]⁻, 1425 [M+K]⁺

(22) E-Cereulide and EAHA-Cereulide (Removal of Benzyl Group byHydrogenolysis Reaction)

A flask for hydrogenation was charged with a mixture of 41 (20 mg),methanol (5 mL) and 10% palladium carbon (2 mg), and the mixture wasstirred under a hydrogen stream (3 atmospheres) at room temperature for3 hours. After confirming the progress of the reaction by TLC, thecatalyst was removed by filtration, and the filtrate was concentratedunder reduced pressure to obtain E-cereulide and EAHA-cereulide.

E-Cereulide;

Yield; quant

TLC; Rf=0.39 (B)

¹H-NMR (CDCl₃); 0.78-1.12 (48H, m), 1.40-1.54 (9H, m), 1.60-1.90 (8H,m), 2.06-2.16 (1H, m), 2.17-2.42 (6H, m), 2.42-2.63 (3H, m), 4.06-4.15(2H, m), 4.25-4.35 (1H, m), 4.35-4.43 (1H, m), 4.43-4.56 (2H, m),4.70-4.82 (1H, m), 4.92-5.04 (1H, m), 5.13-5.31 (1H, m), 6.82 (1H, s),7.60-7.92 (6H, m), 5.07 (1H, d, J=3.17 Hz), 5.09-5.15 (1H, m), 5.17-5.25(1H, m), 5.25-5.33 (1H, m), 7.34 (1H, d, J=5.85 Hz), 7.43-7.50 (1H, m),7.60-7.82 (4H, m), 8.03 (1H, s)

¹³C-NMR (CDCl₃); 16.28, 16.48, 16.65, 16.85, 16.94, 18.52, 18.56, 18.73,19.05, 19.17, 19.24, 21.08, 21.34, 21.40, 21.52, 23.08, 23.22, 23.29,24.39, 24.61, 24.64, 27.93, 28.73, 29.70, 30.12, 30.48, 40.44, 40.50,48.26, 48.46, 48.57, 52.48, 59.42, 72.71, 72.88, 73.57, 77.22, 77.92,78.22, 78.53, 170.00, 170.11, 170.84, 171.16, 171.22, 171.34, 171.45,171.54, 171.95

IR; 3435, 3316, 2964, 1742, 1654, 1538, 1466, 1383, 1198

MALDI/TOFMS; 1205 [M+Na]⁻, 1221 [M+K]⁺

EAHA-Cereulide;

Yield; quant

TLC; Rf=0.32 (B)

¹H-NMR (CDCl₃); 0.75-1.00 (42H, m), 1.06 (6H, d, J=5.85 Hz), 1.34-1.58(13H, m), 1.58-1.86 (11H, m), 1.95-2.10 (1H, m), 2.17-2.42 (10H, m),3.09-3.26 (1H, m), 3.26-3.44 (1H, m), 4.00-4.12 (1H, m), 4.12-4.22 (1H,m), 4.27-4.40 (2H, m), 4.40-4.50 (2H, m), 4.97 (1H, d, J=3.17 Hz),4.99-5.08 (2H, m), 5.13-5.31 (1H, m), 6.82 (1H, s), 7.60-7.92 (6H, m)

IR; 3304, 2963, 2938, 2875, 1743, 1655, 1539, 1466, 1373, 1197

MALDI/TOFMS; 1319 [M+Na]⁻, 1335 [M+K]⁺

1. A precursor of a cereulide derivative, selected from the groupconsisting of the following formulae:

wherein Y represents OH or NH(CH₂)₅COOH.
 2. A didepsipeptide representedby

wherein X represents (CH₂)₂COOH or (CH₂)₂CONH(CH₂)₅COOH.
 3. Adepsipeptide as shown below

wherein Y represents OH or NH(CH₂)₅COOH, l is an integer of 0 to 2, n isan integer of 0 to 2, m is 1, with the proviso that l, m and n are notsimultaneously 0, and l+m+n is 2 or less.
 4. A method for producing acereulide derivative represented by

wherein X represents (CH₂)₂COOH or (CH₂)₂CONH(CH₂)₅COOH, comprising acyclization reaction by formation of an intramolecular amide bond of theprecursor of a cereulide derivative as defined in claim
 1. 5. Theproduction method according to claim 4, comprising a step of preparing adidepsipeptide represented by

wherein X represents (CH₂)₂COOH or (CH₂)₂CONH(CH₂)₅COOH.
 6. Theproduction method according to claim 5, further comprising a step ofpreparing a depsipeptide as shown below

wherein Y represents OH or NH(CH₂)₅COOH, l is an integer of 0 to 2, n isan integer of 0 to 2, m is 1, with the proviso that l, m and n are notsimultaneously 0, and l+m+n is 2 or less.
 7. A cereulide derivativerepresented by the following formula:

wherein R represents (CH₂)₂COOH or (CH₂)₂CONH(CH₂)₅COOH.